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Thrombin receptor activation results in calcium signaling and protein kinase C-dependent stimulation of DNA synthesis in HEp-2g laryngeal carcinoma cells
Abstract
Recently, the expression of the "tethered ligand" thrombin receptor in carcinosarcoma and melanoma cells has been shown. However, the role of the thrombin receptor in tumor cell metabolism still is undefined.
In this article, the "tethered ligand" thrombin receptor was identified on human epidermoid carcinoma cells (HEp-2g cell line) by using immunofluorescence studies with a monoclonal antithrombin receptor antibody and radioligand binding. Furthermore, the effects of alpha-thrombin and thrombin receptor activating peptides (TRAP)-6 on calcium mobilization, protein kinase C (PKC) translocation, and DNA synthesis were estimated.
Pharmacologic characterization using [3H]TRAP-6 as a radioligand demonstrated a single class of high affinity binding sites (dissociation constant [KD] = 7.2 +/- 2.2 x 10(-7) M) and a binding capacity of 27 +/- 3.4 fmol/mg protein. The function of these binding sites was demonstrated by alpha-thrombin- and TRAP-6-induced mobilization of free intracellular calcium, and translocation of PKC from cytosol to cell membrane. Moreover, alpha-thrombin and TRAP-6 induced an increase in [3H]thymidine incorporation in HEp-2g cells that could be blocked by the PKC inhibitor bisindolylmaleimide.
To the authors' knowledge, the results of this study demonstrate for the first time functional thrombin receptors in epidermoid carcinoma cells. The thrombin receptor appears to be involved in growth regulation in HEp-2g cells by a PKC-dependent mechanism.
... Fibronectin (Bd Biosciences, San Jose, CA, USA) was diluted to a final concentration of 100 µg/ml in PBS and 40 µl was added to each well of 96-well plates at 4˚C overnight. Hep-2 cells were pretreated with various concentrations of rH (25,50 or 100 µg/ml) or doX (4 µg/ml) for 0.5 h. After treatment, 1x10 5 cells were added to each well of 96-well plates coated with fibronectin and incubated for 2 h. ...
... A Transwell assay was performed using polycarbonate Transwell filters (Corning Costar, Cambridge, MA, USA). Briefly, cells were suspended in culture medium containing various concentrations of rH (25,50 or 100 µg/ml) or doX (4 µg/ml) and plated to the upper chamber with the bottom filled with culture medium containing basic fibroblast growth factor (bFGF) (Roche, Basel, Switzerland) (3 ng/ml). After incubation for 24 h, the cells on the upper side of the filters were removed mechanically, while cells that migrated to the bottom side were fixed in 4% (w/v) paraformaldehyde. ...
... The upper surface of the filter was coated with 60 µl Matrigel (4 mg/ml) at 37˚C for 30 min. Cells were suspended in culture medium containing various concentrations of rH (25,50 or 100 µ/ml) or doX (4 µg/ml) and added to the upper chamber. The bottom chambers were filled with culture medium containing bFGF (3 ng/ml). ...
Recombinant hirudin (rH) is a highly potent and specific inhibitor of thrombin, and has been shown to inhibit the growth and metastasis of several types of cancers in experimental tumor models. The objective of this study was to evaluate the antitumor effects and explore the underlying mechanisms of rH in Hep-2 human laryngeal carcinoma (LC) cells. Hep-2 cells were treated with various concentrations of rH for 24 h. The cell viability was evaluated by a water-soluble tetrazolium salt (WST) assay. The adhesion ability of the cells was evaluated by cell adhesion to fibronectin. Cell migration and invasion were measured with the Boyden chamber assay. Cell apoptosis was detected by Hoechst 33324 fluorescence staining. A chicken chorioallantoic membrane (CAM) assay was used to assess the effects of rH on angiogenesis in vivo. Western blotting was used to detect the expression levels of vascular endothelial growth factor receptor (VEGF-R), focal adhesion kinase (FAK), Bcl-2-associated agonist of cell death (Bad) and B-cell CLL/lymphoma 2 (Bcl-2) proteins. rH significantly inhibited the cell viability and induced apoptosis in LC Hep-2 cells in a dose-dependent manner, as compared with phosphate-buffered saline (PBS) as control. These results were accompanied by a decrease in the anti-apoptotic protein Bcl-2 and an increase in the pro-apoptotic protein Bad. Moreover, rH dose-dependently inhibited the adhesion, migration and invasion of the Hep-2 cells, compared to the vehicle PBS. In addition, rH robustly suppressed angiogenesis in the CAM assay. Importantly, the expression of adhesion and angiogenesis-associated proteins FAK and VEGF-R was significantly downregulated by rH in a dose-dependent manner. The present findings demonstrate that rH exerts antitumor effects in Hep-2 human laryngeal cancer cells via multiple mechanisms and suggests that targeting thrombin by rH is a potential strategy for the treatment of LC.
... In vitro experiments showed that activation of TR increased the proliferation rate of osteoblasts and stimulated the activity of alkaline phosphatase (ALP), an osteoblast differentiation marker [14]. TR has been reported to convey its intracellular signal via the PLC-β, p42/44-ERK, and PKC pathways [15,16]. By using in vitro calvarial and long bone cultures, thrombin induced bone resorption, while TR knockout mice (TR KO) manifested decreased migration and proliferation of mesenchymal cells into the bone fracture area of bone-drilled model [11,17,18]. ...
... The serine proteases can exert their physiological effects via various signaling pathways including p42/44-ERK, p38-MAPK, PKC, and Rac1 [15,16,31,45,46]. Upon stimulation by thrombin; however, TR conveys its signal via either PKC through G q11 /G i2 or p42/44-ERK through G 12 / G 13 [16,[47][48][49][50]. ...
... The serine proteases can exert their physiological effects via various signaling pathways including p42/44-ERK, p38-MAPK, PKC, and Rac1 [15,16,31,45,46]. Upon stimulation by thrombin; however, TR conveys its signal via either PKC through G q11 /G i2 or p42/44-ERK through G 12 / G 13 [16,[47][48][49][50]. Based on our finding that thrombin stimulated RANKL expression through p42/44 ERK signaling, it was thus presumed in the present study that TR mediated its effect via G 12 /G 13 . ...
Thrombin and its receptor (TR) are, respectively, expressed in osteoclasts and osteoblasts. However, their physiological roles on bone metabolism have not been fully elucidated. Here we investigated the bone microarchitecture by micro-computed tomography (μCT) and demonstrated increased trabecular and cortical bone mass in femurs of TR KO mice compared to WT littermates. Trabecular thickness and connectivity were significantly enhanced. The physiological role of TR on both inorganic and organic phases of bone is illustrated by a significant increase in BMD and a decrease in urinary deoxypyridinoline (DPD) crosslink concentration in TR KO mice. Moreover, TR KO cortical bone expanded and had a higher polar moment of inertia (J), implying stronger bone. Bone histomorphometry illustrated unaltered osteoblast and osteoclast number and surface in femoral metaphyses, indicating that thrombin/TR regulates osteoblasts and osteoclasts at functional levels. Serum analysis showed a decrease in RANKL and an increase in osteoprotegerin (OPG) levels and reflected a reduced RANKL/OPG ratio in the TR KO group. In vitro experiments using MC3T3 pre-osteoblasts demonstrated a TR-dependent stimulatory effect of thrombin on the RANKL/OPG ratio. This effect was blocked by TR antagonist and p42/p44-ERK inhibitor. In addition, thrombin also intensified p42/p44-ERK expression and phosphorylation. In conclusion, the thrombin/TR system maintains normal bone remodeling by activating RANKL and limiting OPG synthesis by osteoblasts through the p42/44-ERK signaling pathway. Consequently, TR deficiency inhibits osteoclastogenesis, resulting in a high bone mass phenotype.
... The ability of thrombin to act via PARs was highlighted by the demonstration of the ability of PAR 1 to stimulate tumour invasion [ 53,54 ] by its expression in carcinosarcoma and melanoma cells [ 55 ] . The extensive work in this fi eld related to tumour tissue done over the past decade has therefore focused primarily on PAR 1 for which the expression and signaling at the cellular level have been characterized in tumour cells from different tumour entities including cancers of the larynx [ 56 ] , pancreas [ 57 ] , glioma [ 58,59 ] , glioblastoma [ 60,61 ] , meningioma [ 62 ] , prostate [ 63 ] and colon [ 64 ] . In addition, PAR 1 activation has been observed to cause (I) increased tumour cell adhesion to the endothelium, extracellular matrix and platelets, (II) enhanced metastatic capacity of tumour cells, (III) activated cell growth and (IV) increased angiogenesis [65][66][67] . ...
... The presence of a specifi c PAR in a target cancer cell and its ability to increase intracellular calcium can be established using a receptor cross-desensitization protocol with PAR-selective agonists and appropriate PAR-inactive 'control' peptides [ 126 ] . This approach that uses fl uorimetric methods to monitor calcium transients with different calcium sensitive fl uorescence dyes has documented PAR-mediated increases in [Ca 2+ ] i in cells from various malignancies including those from brain [ 53,56,57,109 ] , colon [ 64,74 ] , pancreas [ 127 ] , kidney [ 128 ] , breast [ 19 ] , larynx [ 56 ] , prostate [ 112 ] and liver [ 72 ] . Although all of PARs 1, 2 and 4 can couple with G q to elevate intracellular calcium in all PAR-expressing cells so far examined, the precise downstream consequences of elevated calcium per se have not been established in any detail. ...
... The presence of a specifi c PAR in a target cancer cell and its ability to increase intracellular calcium can be established using a receptor cross-desensitization protocol with PAR-selective agonists and appropriate PAR-inactive 'control' peptides [ 126 ] . This approach that uses fl uorimetric methods to monitor calcium transients with different calcium sensitive fl uorescence dyes has documented PAR-mediated increases in [Ca 2+ ] i in cells from various malignancies including those from brain [ 53,56,57,109 ] , colon [ 64,74 ] , pancreas [ 127 ] , kidney [ 128 ] , breast [ 19 ] , larynx [ 56 ] , prostate [ 112 ] and liver [ 72 ] . Although all of PARs 1, 2 and 4 can couple with G q to elevate intracellular calcium in all PAR-expressing cells so far examined, the precise downstream consequences of elevated calcium per se have not been established in any detail. ...
Proteinase activated receptors (PARs), a small subfamily of G protein-coupled receptors with four members, PAR₁, PAR₂, PAR₃ and PAR₄, are expressed in various tumours from epithelial origin and can play an important role in tumour progression and metastasis. Within the complex intracellular PAR signaling networks triggered by PARs, an elevation in intracellular free calcium ion concentrations represents a key second messenger system. In this review, we summarize current information about the mechanisms whereby PARs can signal via intracellular calcium in the setting of cancer and we discuss possibilities for using the PAR-[Ca(2+)](i) signaling pathway as a target for the therapy of epithelial cancer.
... Protease-activated receptor-1 (PAR-1), the prototypic member of the PAR family, is activated by thrombin following cleavage of its extracellular amino terminus domain (1)(2)(3)(4)(5)(6)(7). PAR-1 and its activating factors, which are expressed on tumor cells and their stroma, induce coagulation and have a significant role in promoting tumor progression in several carcinomas such as breast, pancreas, laryngeal and gastric cancer (1,7). ...
... Protease-activated receptor-1 (PAR-1), the prototypic member of the PAR family, is activated by thrombin following cleavage of its extracellular amino terminus domain (1)(2)(3)(4)(5)(6)(7). PAR-1 and its activating factors, which are expressed on tumor cells and their stroma, induce coagulation and have a significant role in promoting tumor progression in several carcinomas such as breast, pancreas, laryngeal and gastric cancer (1,7). ...
Protease-activated receptor-1 (PAR-1) has a significant role in the pathogenesis of various malignancies and its expression mainly affects the survivals of cancer patients. The aim of the present study was to determine the clinical significance of the serum concentrations of PAR-1 in patients with gastric carcinoma. A total of 63 pathologically confirmed gastric cancer patients were enrolled in this study, with a median age of 62 years. Serum PAR-1 concentrations were determined by the enzyme-linked immunosorbent assay method and no significant difference in the baseline serum PAR-1 concentrations was found between patients and normal controls (P=0.5). The investigated clinical variables, including patient age, gender, localization of lesion, histology, grade of pathology, disease stage and serum tumor markers (lactate dehydrogenase, carcinoembryonic antigen and carbohydrate antigen 19-9) were not correlated with serum PAR-1 levels (P>0.05). Furthermore, no association was identified between the serum PAR-1 level and chemotherapy responsiveness (P=0.43). Serum PAR-1 level also had no prognostic role for survival (P=0.27). In conclusion, the serum PAR-1 concentration has no diagnostic, predictive and prognostic values in gastric cancer patients.
... Synthetic peptides of 5-14 amino acids (thrombin receptor activating peptides, TRAP's), corresponding to the tethered ligand sequence, have been found to be agonists for receptor activation [1]. Besides in a variety of non-neoplastic cells, functional PAR 1-type thrombin receptors have been identified in different tumor cell lines [6][7][8][9] suggesting a role of PAR 1 concerning thrombin-induced effects in tumors including promotion of tumor cell adhesion to endothelium and extracellular matrix as well as enhancement of metastatic capacity and growth of tumors [10][11][12]. ...
Thrombin is known to play a role as regulator in tumor spreading and tumor growth. Proteinase-activated receptor 1 (PAR 1)-type thrombin receptors were identified in different cancer cells including human glioblastoma cells. Thus a function of PAR 1 in brain tumors may be suggested. In this study, the presence of PAR 1-type thrombin receptors was investigated in primary cell cultures established from operated human meningiomas from two 59- and 79-year-old women. Characterization of PAR 1 on binding level was performed using immunofluorescence studies with the monoclonal anti-PAR 1 antibody Mab 61-1 directed against a domain in the NH2-terminus of PAR 1. These binding sites constitute functional thrombin receptors that are involved in thrombin-induced signaling in human meningioma cells as demonstrated by investigation of alpha-thrombin- and PAR 1-activating hexapeptide (TRAP-6)-induced [Ca2+]i mobilization. To our knowledge, this is the first report demonstrating thrombin-induced intracellular signaling in human meningioma cells mediated by the PAR 1-type thrombin receptor.
... Investigation of the role of PAR-1 in tumorigenesis and metastasis was initiated only recently. PAR-1 expression has been detected in human colon adenocarcinoma [9], a pancreatic tumor cell line [10], and a laryngeal carcinoma cell line [11]. Furthermore, two laboratories have demonstrated that PAR-1 is expressed at higher levels in highly metastatic breast cancer cell lines than breast cancer cell lines with low metastatic potential [12,13]. ...
Protease-activated receptor 1 (PAR-1) is a G-coupled membrane protein. In this study, we analyzed the expression of PAR-1 in oral squamous cell carcinomas (SCCs). PAR-1 was expressed in oral SCCs, but the level of PAR-1 protein was lower in non-metastatic cells than in metastatic cells. Thrombin stimulated the growth of metastatic cells, and both thrombin and thrombin receptor activation peptide (TRP) enhanced the adhesion of these cells to fibronectin, but had no effect on non-metastatic cells. Thrombin and TRP also induced matrix metalloproteinase (MMP)-2 and MMP-9 activities in metastatic cells. These results suggest that PAR-1 may contribute to the growth and invasive potential of oral SCC.
... PAR-1 expression has been detected in human melanoma (12), colon adenocarcinoma (13), pancreatic cancer (14), and SCCHN (15,16). However, the role of PAR-1 in metastasis has not been clearly defined in current publications. ...
Protease-activated receptor-1 (PAR-1) is a G-protein-coupled receptor that contributes to multiple signal transduction pathways. Although the functions of PAR-1 in many normal cells, such as platelets and astrocytes, have been well studied, its roles in cancer progression and metastasis have not been fully elucidated, and studies to date appear contradictory.
To clarify the function of PAR-1 in metastasis of squamous cell carcinoma of the head and neck (SCCHN), we examined PAR-1 expression in clinical specimens by immunohistochemistry and in SCCHN cell lines by immunoblotting. Furthermore, par-1 cDNA-transfected SCCHN cell lines were also used to verify PAR-1-mediated pathway.
The metastatic tumors showed a lower percentage of PAR-1-positive cells (46%) and lower levels of PAR-1 expression (median weight index = 10) than node negative primary tumors (80% and median weight index = 60, respectively). In addition, expression level of PAR-1 positively correlated with levels of keratinocyte differentiation markers keratin-1, -10, and -11. Additional studies using sense and antisense par-1 cDNA-transfected SCCHN cell lines illustrated that the presence of PAR-1 was required for the expression of involucrin, a keratinocyte differentiation marker. PAR-1 expression also contributes to activation of the mitogen-activated protein kinase (MAPK) pathway. Blocking MAPK activation by a mitogen-activated protein/extracellular signal-regulated kinase inhibitor, not by a phosphatidylinositol 3'-kinase inhibitor, reduced level of involucrin, suggesting that regulation of involucrin by PAR-1 is partially through the MAPK signaling pathway.
Our study suggests that PAR-1 signaling induces differentiation markers in SCCHN cells, and its expression is conversely correlated with cervical lymph node metastasis.
... Obviously, thrombin stimulation of tumor growth requires the participation of Ras protein, protein kinase C (PKC), and the mitogen-activated protein kinase (MAPK) pathway [47]. Consequently, mitogenic activities of thrombin and PAR-1 activation could be abolished by the PKC inhibitor bisindolylmaleimide in human epidermoid carcinoma cells [48]. Also, the MEK inhibitor PD98059 suppressed thrombininduced cell proliferation in human colon cancer cells [49]. ...
The activation of the coagulation system in cancer patients is a well-known phenomenon responsible for recurrent clinical problems. A number of fascinating molecular mechanisms have been recognized showing that the tumor not only activates the coagulation system, but vice versa, activated coagulation proteins are able to induce molecular effects in tumor cells. The molecular basis is the expression of defined membrane receptors by tumor cells that are activated, for example, by thrombin. As the liberation of thrombin from prothrombin is one of the key events in coagulation, it's impact upon biological processes, such as cancerogenesis and metastasation, seems to be a regular pathophysiological consequence. These perceptions are not only interesting for the comprehension of cancerogenesis, metastasation, and clinical phenomena, but they also have a high impact upon modern strategies of tumor therapy. Especially, the development of clinically useful coagulation inhibitors, such as modern low molecular weight heparins or melagatran, created the possibility of therapies that combine cell biological approaches with apoptosis-inducing principals such as chemotherapy. Several clinical studies that demonstrate the implication of these strategies have already been published recently. In this article the cell biological basics for these approaches are reviewed.
... There is emerging evidence that PAR-1 modulates cell proliferation and motility in physiopathologic cell invasion processes, suggesting that it plays a role in the setting of cancer growth and metastasis [9][10][11][12][13]. Indeed, overexpression of PAR-1 has been detected in numerous human cancers, including colon [14,15], laryngeal [16], breast [10], pancreatic [17,18], and oral cavity carcinomas [19]. Of interest, an up-regulation of PAR-1 and PAR-2 has been demonstrated in stromal fibroblasts surrounding neoplastic aggregates in human malignant tissues [20]. ...
Protease-activated receptors (PARs) are members of the G protein-coupled receptor superfamily that are activated by the proteolytic cleavage of their amino terminal domain. PAR-1 activation by thrombin results in several biologic effects, including platelet adhesion to other cells or extracellular matrix, fibroblast, and endothelial cell growth, whereas PAR-2, activated by trypsin, has mainly a proinflammmatory and angiogenetic role. PAR-1 and PAR-2 modulate cell proliferation in physiopathologic cell invasion processes, suggesting that they may play a role in the setting of cancer growth and metastasis. Here, we have investigated the expression of PAR-1 and PAR-2 proteins by immunohistochemistry in a series of benign and malignant melanocytic lesions: 20 melanocytic lesions (10 common melanocytic nevi and 10 atypical or "dysplastic" melanocytic nevi) and 50 melanomas (10 in situ melanomas, 10 melanomas T1, 10 melanomas T2, 10 melanomas T3 to T4, and 10 metastatic melanomas). PAR-1 was significantly overexpressed in atypical nevi and melanomas in comparison with common melanocytic nevi. PAR-2 was strongly and diffusely expressed by immunohistochemistry in all melanocytic lesions, with no statistically significant differences between nevi and melanomas. Because we found a differential expression in PAR-1 protein, but not in PAR-2, we next investigated the expression of PAR-1 messenger RNA (mRNA) by ribonuclease protection assay in paraffin-embedded tissues using a paraffin block RNA isolation procedure. Similarly to immunohistochemical results, PAR-1 mRNA expression was significantly higher in atypical nevi and melanomas in comparison with common nevi and controls. Overexpression of PAR-1 in atypical nevi and melanomas supports a role for PAR-1 in the initial phases of melanoma development as well as in tumor progression and metastasis. Conversely, the significance of PAR-2 up-regulation in both benign and malignant melanocytic lesions requires further research.
... Wojtukiewicz et al demonstrated the expression of PAR 1 in carcinosarcoma and melanoma cells (10) suggesting a role of thrombin receptors in carcinogenesis. During the following years, thrombin receptors were characterized on signaling and cellular level in cells from different tumor entities including larynx (11), pancreas (12), brain (13), prostate (14), breast (15) and colon (16). The very extensive work done in this field over the past 5-10 years demonstrates for cancer that thrombin can signal via a diverse set of mechanisms and there is no single signal transduction pathway activated by thrombin in all situations. ...
Cross-talk between G-protein-coupled receptor (GPCR) and epidermal growth factor receptor (EGFR) signaling systems is established in a wide variety of normal and neoplastic cell types. Here, we show that proteinase-activated receptor 1 (PAR1) mediates the tyrosine phosphorylation of EGFR in human renal carcinoma cells expressing PAR1 and PAR3 endogeneously. This GPCR-EGFR signal transduction pathway cross-talk requires matrix metalloproteinase activity and is involved in the regulation of renal carcinoma cell migration across a collagen barrier as shown using a Boyden chamber type assay. Our data therefore document a regulatory role of PAR1-mediated EGFR transactivation in cancer cell chemotactic migration. Further, our results underline the importance of PAR1-mediated pathways in kidney cancer cells and suggest that the thrombin/PAR1 system mediating EGFR transactivation may play a role in the progression of this tumor entity.
... PARs constitute a family of G proteincoupled receptors that are enzymatically activated by serine proteases through cleavage of their amino terminal domain, and their activation is implicated in numerous biological effects, including inflammation (Asokananthan et al., 2002), coagulation (Coughlin, 2001;Ruf et al., 2003), mitogenesis (Madamanchi et al., 2001), and cell proliferation (Darmoul et al., 2003). Overexpression of the protease-activated receptor-1 (PAR-1) has been reported in a variety of human carcinoma cell lines, including colon (Wojtukiewicz et al., 1995), laryngeal (Kaufmann et al., 1997), breast (Even-Ram et al., 1998;Henrikson et al., 1999), pancreatic (Rudroff et al., 1998), and oral squamous cell neoplasms (Liu et al., 2001). In cutaneous melanomas, we observed a correlation between the expression of PAR-1 and the metastatic potential of human melanoma cell lines . ...
The identification of molecular markers of melanoma progression is needed to more accurately stage and identify treatments for patients with malignant melanoma. Previously, we demonstrated that loss of the activator protein-2alpha (AP-2alpha) expression results in overexpression of the protease-activated receptor-1 (PAR-1) in human melanoma cell lines. Here, we used a tissue microarray platform that consisted of 64 melanocytic lesions, including dysplastic nevi (N=21), primary melanoma (N=20), and metastatic melanoma (N=23). We analyzed the expression of AP-2 and PAR-1 simultaneously by immunofluorescent microscopy with an automated quantification laser scanning cytometer. AP-2 was highly expressed in normal cutaneous melanocytes and dysplastic nevi but not in melanoma metastases. We observed a significantly higher number of AP-2-positive cells in the dysplastic nevi (P=0.0013) and primary melanoma (P=0.0023) compared to the metastatic melanoma. In contrast, we observed a significantly higher percentage of PAR-1-positive cells in the metastatic melanoma compared to dysplastic nevi (P=0.0072) and primary melanoma (P=0.0138). Increased expression of PAR-1 in metastatic melanomas contributes to tumor progression by modulating expression of genes, such as IL-8, matrix metalloproteinase-2, vascular endothelial growth factor, platelet-derived growth factor, and integrins. These findings support our hypothesis that loss of AP-2 is a crucial event in the progression of human melanoma and contributes to the acquisition of the metastatic phenotype via upregulation of PAR-1.
... decade has focused primarily on PAR 1 , with little attention yet paid to a potential role for PAR 4 . PAR 1 expression and signaling at the cellular level have been characterized for a wide range of tumor cells (Kaufmann et al., 1997;Even-Ram et al., 1998;Chay et al., 2002;Darmoul et al., 2003) and other cell types present in the tumor microenvironment (D'Andrea et al., 2001). At present there is substantial evidence that thrombin acting via PAR 1 contributes to the metastatic process of certain epithelial tumors including breast (Even-Ram et al., 1998Henrikson et al., 1999), colon (Darmoul et al., 2003), and kidney (Bergmann et al., 2006). ...
Proteinase-activated receptor-1 (PAR(1)), a thrombin receptor and the prototype of a newly discovered G-protein-coupled receptor subfamily, plays an important role in tumor development and progression. In this study, we documented the expression of the thrombin receptors PAR(1), PAR(3), and PAR(4) in permanent hepatocellular carcinoma (HCC) cell lines and primary HCC cell cultures. Stimulation of HCC cells with thrombin and the PAR(1)-selective activating peptide, TFLLRN-NH(2), increased transmembrane migration across a collagen barrier. This effect was blocked by the PAR(1) antagonist SCH 79797, confirming that the PAR(1) thrombin receptor subtype is involved in regulating hepatoma cell migration. In addition, the PAR(4)-selective agonist, AYPGKF-NH(2), also stimulated HCC cell migration whilst the PAR(4) antagonist, trans-cinnamoyl-YPGKF-NH(2), attenuated the effect of thrombin on HCC cell migration. PAR(1)- and PAR(4)-triggered HCC cell migration was blocked by inhibiting a number of key mediators of signal transduction, including G proteins of the G(i)/G(o) family, matrix metalloproteinases, ERK/MAPKinase, cyclic AMP-dependent protein kinase, Src tyrosine kinase, and the EGF receptor kinase. Our data point to a cooperative PAR(1)/PAR(4) signaling network that contributes to thrombin-mediated tumor cell migration. We suggest that a combined inhibition of coagulation cascade serine proteinases, the two PARs and their complex signaling pathways may provide a new strategy for treating hepatocellular carcinoma.
... cancers, PAR-1 correlates with tumor progression by augmenting the expression of angiogenic factors such as vascular endothelial growth factor (VEGF) [30] and, in prostate cancers, it has been implicated in bone metastasis [31]. Indeed, PAR-1 has been reported to be highly expressed in pancreatic adenocarcinoma [32], laryngeal carcinoma [33], renal carcinoma [34], lung adenocarcinoma, gastric carcinoma [8], and oral squamous cell carcinoma [35]. This is the first study to demonstrate the role of PAR-2 in ovarian cancers, to the best of our knowledge. ...
Protease activated receptor-2 (PAR-2) has been implicated in cellular proliferation, invasion and metastasis with angiogenesis in various tumors. This prompted us to study the role of PAR-2 in tumor advancement of ovarian cancers.
Forty-eight patients underwent surgery for ovarian cancers. In ovarian cancers, PAR-2 histoscores and mRNA levels were determined by immunohistochemistry and real-time reverse transcription-polymerase chain reaction, respectively. Patient prognosis was analysed with a 36-month survival rate. Microvessel counts were determined by immunohistochemistry for CD31 and factor VIII-related antigen and the rate of cell proliferation was determined by immunohistochemistry for Ki67.
Immunohistochemical staining revealed distribution of PAR-2, dominantly in cancer cells and faintly in stromal cells of the tumor. PAR-2 histoscores in cancer cells and mRNA levels both significantly increased in ovarian cancers with clinical stages (I < II < III < IV, P < 0.05), regardless of histopathological type. The 36-month survival rate of 24 patients with high PAR-2 was poor (58%), while that of the other 24 patients with low PAR-2 was significantly higher (83%). There were significant correlations between PAR-2 histoscores in cancer cells and mRNA levels with microvessel counts and with the rate of cell proliferation in ovarian cancers.
PAR-2 was up-regulated during ovarian cancer progression. Therefore, PAR-2 might work on tumor advancement of ovarian cancers via angiogenic activity and is considered to be a novel prognostic indicator in ovarian cancers.
Aim:
This study aimed to analyze the predictive, prognostic and diagnostic value of autoantibodies to coagulation factor II thrombin receptor (F2R; protease-activated receptor 1, PAR1) (PAR1-AB) in patients with primary epithelial ovarian cancer (EOC).
Materials and methods:
A total of 197 patients with primary EOC and 200 healthy female blood donors were included in the study. Enzyme-linked immunosorbent assay was applied to determine PAR1-AB levels in blood sera taken preoperatively. Correlation of PAR1-AB with clinicopathological outcome, progression-free (PFS) and overall (OS) survival was analyzed and patients were compared with controls.
Results:
PAR1-AB was significantly negatively correlated with histological grading (p=0.008) and was significantly lower in the patient group compared to healthy controls (p<0.001). There was no significant correlation of PAR1-AB level with PFS or OS.
Conclusion:
This study showed PAR1-AB to significantly decrease in patients with primary EOC and with histological high-grade carcinoma. The relevance of PAR1-AB in early detection of ovarian cancer and follow-up for EOC should be further investigated.
PAR(proteinase-activated receptor)-type thrombin receptors are members of a novel family of seven-transmembrane G-protein coupled receptors. The discovery of this receptor subclass has provided a framework for understanding how the coagulation proteinase thrombin communicates with cells. Recent studies provided evidence for the crucial role of PARs in tumor development and progression. Renal carcinomas belong to a coagulation type tumors where the tumor cells are associated with intact coagulation pathway that leads to thrombin formation. Therefore, investigation of PARs in this tumor entity is highly important on the definition of their significance in malignancy. In this chapter, the present knowledge of PAR-type thrombin receptors in renal carcinoma cells is summarized. After an overview including PAR structure and their effects in different tumor cell types; PAR characterization on RNA, protein, signaling and cellular level in renal carcinoma cells (permanent human renal carcinoma cell line A-498 and primary cultures obtained from surgically resected renal cell carcinomas) is described. A potential role of PAR-type thrombin receptors in renal cell carcinoma prognosis and therapy is discussed.
Results from clinical studies indicate a strong association between thromboembolism and solid malignancy. Being central to blood coagulation and displaying a number of cellular postclotting activities, the serine protease thrombin has been localized within or adjacent to malignant tissues possibly associated with fibrin(oid) deposits. Thrombin proteolytically activates its cellular receptors including the prototypic protease-activated receptor I which is expressed by various tumor cells. Current molecular and cellular studies provide mounting evidence that the activation of protease-activated receptor(s) is the main mechanism whereby thrombin exerts its modulating effects on the malignant phenotype including tumor growth, local progression, and distant metastasis. A detailed understanding of the molecular interplay between thrombin, thrombin receptor(s) and cancer biology may be helpful to develop new therapeutic approaches consisting of the suppression of thrombin receptor(s) in malignancy.
To determine treatment strategies and predict the clinical outcome of patients with melanoma it is important to understand
the etiology of this disease. Recently, there has been some insight into the molecular basis of melanoma, including identification
of a few of the regulatory factors and genes involved in this disease. For instance, the transcription factor, activator protein
(AP)-2α, plays a tumor suppressor- like role in melanoma progression by regulating genes involved in tumor growth and metastasis.
Previously, we have shown that the progression of human melanoma to the metastatic phenotype is associated with loss of AP-2
expression and deregulation of target genes, such as MUC18/MCAM, c-KIT, and MMP-2. This chapter focuses on the expression of the thrombin receptor (protease-activated receptor [PAR]-1 ) in human melanoma
and its regulation by AP-2 and specificity protein (Sp)-1. We demonstrate that the metastatic potential of human melanoma
cells correlates with overexpression of PAR-1. We also provide evidence that an inverse correlation exists between the expression
of AP-2 and the expression of PAR-1 in human melanoma cells. The regulatory region of the PAR-1 gene contains multiple AP-2 consensus elements overlapping multiple Sp1 consensus elements. Our analysis of the highly overlapping
AP-2 and Sp1 binding elements (complex 1, bp -365 to -329) within the regulatory region demonstrates that AP-2 and Sp1 bind
to this region in a mutually exclusive manner to promote repression or activation, respectively. We propose that loss of AP-2
results in increased expression of the thrombin receptor, which subsequently contributes to the metastatic phenotype of melanoma
by upregulating the expression of adhesion molecules, proteases, and angiogenic molecules.
Malignant progression and tumor metastasis is a complex process enabled by various molecular changes occurring in a subpopulation
of tumor cells. The metastatic phenotype is associated with the cellular capacity for uncontrolled growth, resistance to apoptosis,
high invasive potential, and effective neoangiogenesis. Whereas the contribution of genetic alterations to the metastatic
dissemination is not yet clear, because both primary and metastatic tumors often have similar patterns of genetic mutations,
the majority of the changes contributing to the metastatic phenotype are controlled epige-netically. In melanoma, the progression
toward malignant disease and acquisition of the metastatic phenotype involves loss of activator protein 2 and gain in expression
of activating transcription factor 1/cyclic adenosine monophosphate-responsive element-binding protein family transcription
factors. Together with upregulation of activating transcription factor 2, Snail, nuclear factor-?B and other transcription
factors, this results in deregulation of the expression of cellular adhesion molecules, matrix-degrading enzymes, as well
as other factors that enable a complex interaction of tumor cells with extracellular milieu and other cells during malignant
progression and metastatic dissemination. Furthermore, because of the need to survive mechanical and immunological challenges,
and changing nutritional environment during the dissemination process, metastatic cells are permanently selected for the superior
survival capacity. As a result, metastatic cells are commonly characterized by their increased resistance to the chemotherapeutic
treatment when compared to primary tumors. Here, we discuss some of the potential mechanisms contributing to drug resistance
in melanoma.
Protease-activated receptor 1 (PAR-1) is a G-coupled membrane protein, which is involved in physiological and malignant invasion processes. It is activated by serine proteases such as thrombin through a unique form or by specific synthetic peptides. In this study, we determined the expression of PAR-1 in five nasopharyngeal carcinoma (NPC) cell lines with different characteristics of invasiveness and metastasis, and found that the levels of PAR-1 expression were higher in invasive or metastatic cell lines than those in low invasive or metastatic ones. Of the five NPC cell lines, CNE1-LMP1 cells had the highest expression levels of PAR-1, which was mainly distributed at the membrane and in the cytoplasm of tumor cells. Further study showed that the thrombin receptor synthetic activating peptide SFLLRN could stimulate the growth of CNE1-LMP1 cells in a dose-dependent manner. However, thrombin itself had a dual effect on the proliferation of NPC cells. Concentrations of thrombin in the range of 0.1-0.5 U/ml promoted cell growth, but concentrations higher than 0.5 U/ml impaired cell growth. Moreover, thrombin and SFLLRN also enhanced the invasive capabilities of CNE1-LMP1 cells in vitro, and this was partly due to enhancing the activities of MMP-2 and MMP-9. Our findings suggest that PAR-1 may contribute to the growth and invasive potential of NPC cells.
This paper describes the development of galactosidase protease-activated receptor (GPAR) as a recombinant protein obtained by fusion of beta-galactosidase, the extracellular domains of protease-activated receptors (PARs), and a biotin acceptor domain. Used as an immobilized substrate, this protein allows the detection of thrombin in the sub-picomolar range. A comparative analysis for proteolytic cleavage of murine PAR1, PAR2, and PAR3 and human PAR4 was performed, involving mutated and nonmutated GPAR fusion proteins. Thrombin cleaved GPAR1 (2.6 mol(beta-galactosidase)/(mol(thrombin) * min)), GPAR3 (410 mmol(beta-galactosidase)/(mol(thrombin) * min)), and GPAR4 (4.3 mmol(beta-galactosidase)/(mol(thrombin) * min)) specifically at the proteolytic activation site. A second possible cleavage site for thrombin is present in murine PAR1 and PAR3. Trypsin and plasmin cleaved all receptor fusion proteins with little specificity for the activation site, except for a marked preference of trypsin for cleavage at the activation site of GPAR2. Chymotrypsin cleaves GPAR1 at a rate (58 mmol(beta-galactosidase)/(mol(thrombin) * min)) that suggests the possibility of chymotryptic inactivation of PAR1. Elastase may inactivate PAR1 and PAR3, but probably not PAR2 and PAR4. Neither activated protein C nor the plasminogen activators cleave any GPAR fusion protein at considerable rates.
To investigate molecular mechanisms of PAR-1 regulation on intracellular Ca²(+) mobilization in lung giant cell carcinoma cells in vitro and its involvement in tumor metastasis.
Free intracellular Ca²(+) ([Ca²(+)]i) was measured in lung giant cell carcinoma PLA801C and PLA801D cells by confocal microscopy. Sense and anti-sense PAR-1 expression vectors were transfected into PLA801C (C+)and PLA801D(D-) cells, respectively. The effects of PAR-1 expression were investigated by thrombin and TRAP-induced mobilization of [Ca²(+)]i in the C+ and D-cells.
There were significant differences of the mean values of [Ca²(+)]i between PLA801D (59.55) and PLA801C cells (35.46, P < 0.01). The mean [Ca²(+)]i of C+ cells (45.77) was significantly higher than that of its control CV cells (35.46, P < 0.05), and the mean [Ca²(+)]i of D-cells (48.42) was significantly lower than that of its control DV cells (59.55, P < 0.05). The peaks of [Ca²(+)]i of C+ and CV cells were 48.19 ± 9.84 and 45.64 ± 9.87 (P < 0.05) respectively at 80 s and 100 s after thrombin treatment, but were 111.31 ± 25.00 and 52.93 ± 11.21 (P < 0.05) respectively at 60 s after TRAP treatment. The peaks of [Ca²(+)]i of D- and DV cells were 40.71 ± 5.89 and 61.07 ± 21.36 (P < 0.05) respectively at 60 s after thrombin treatment, but were 84.98 ± 11.23 and 102.58 ± 21.48 (P < 0.05) respectively at 40 s after TRAP treatment.
The high metastatic potential of PLA801D and PLA801C may be related to [Ca²(+)]i of the tumor cells. PAR-1 may play an important role in the metastasis of lung giant cell carcinoma cells by up-regulating the intracellular Ca²(+).
Human recombinant erythropoietin (rHuEPO) induces cytosolic free calcium ([Ca2+]i) mobilization, an activation of mitogen-activated protein (MAP) kinase and DNA synthesis in several tissues. We explored the mechanism of rHuEPO-induced [Ca2+]i mobilization and its role in the activation of MAP kinase and DNA synthesis in vascular smooth muscle cells (VSMC).
[Ca2+]i concentrations were measured by fura-2. MAP kinase activation was analyzed using an immunocomplex kinase assay and Western blotting. DNA synthesis was measured as an incorporation of 5-bromo-2'-deoxyuridine.
Although addition of rHuEPO significantly increased [Ca2+]i, either in the presence or absence of extracellular Ca2+, the peak level and sustained elevation of [Ca2+]i were significantly reduced in the absence of extracellular Ca2+. Pretreatment with genistein completely blocked the elevation of [Ca2+]i in both conditions. Calphostin C and staurosporine did not completely block the elevation of [Ca2+]i. Staurosporine reduced its peak level in a dose-dependent manner, whereas calphostin C reduced its peak level at concentrations over 1 nmol/l in the presence of extracellular Ca2+. Similar results to those with staurosporine were observed with nifedipine. In the absence of extracellular Ca2+, their dose-dependent effects disappeared even though rHuEPO increased [Ca2+]i. rHuEPO activated MAP kinase and DNA synthesis, both of which were significantly suppressed by the chelation of intracellular Ca2+.
These findings suggest that rHuEPO increases [Ca2+]i by both Ca2+ influx and Ca2+ release from intracellular stores. Tyrosine phosphorylation is critical in the regulation of [Ca2+]i, but protein kinase C activation is important only in the regulation of Ca2+ influx. Dihydropyridine-sensitive L-type Ca2+ channels seem to be involved in rHuEPO-induced Ca2+ influx. In addition, increase of [Ca2+]i by rHuEPO stimulates MAP kinase activation and DNA synthesis in VSMC.
The traditional view on the role of serine proteases in tumor biology has changed with the recent discovery of a family of protease-activated receptors (PARs). In this study we explored the expression and functional role of the thrombin receptor PAR-1 in human colon cancer cells. Reverse transcriptase-polymerase chain reaction analysis showed that PAR-1 mRNAs are present in 11 of 14 human colon cancer cell lines tested but not in normal human colonic epithelial cells. This is in line with the immunolocalization of PAR-1 in human colon tumors and its absence in normal human colonic mucosa. The functional significance of the aberrant expression of PAR-1 in colon cancer cells was then investigated. We found that 1) a prompt increase in intracellular calcium concentration was observed on thrombin (10 nmol/L) or PAR-1 agonist AP1 (100 micro mol/L) challenge of HT29 cells; 2) HT29 quiescent cells treated with thrombin (0.01 to 20 nmol/L) or AP1 (1 to 300 micro mol/L) exhibited dramatic mitogenic responses (3.5-fold increase in cell number). Proliferative effects of thrombin or AP1 were also observed in other colon cancer cell lines expressing PAR-1. This effect was reversed by the MEK inhibitor PD98059 in consonance with the ability of thrombin or AP1 to induce phosphorylation of p42/p44 extracellular-regulated protein kinases. 3) PAR-1 activation by thrombin or AP1 led to a two-fold increase in cell motility of wounded HT29-D4. Our results demonstrate for the first time the aberrant expression of the functional thrombin receptor PAR-1 in colon cancers and its important involvement in cell proliferation and motility. Thrombin should now be considered as a growth factor for human colon cancer.
Evidence for the regulation of cancer growth by components of the blood coagulation mechanism provides abundant opportunity for the development of novel hypotheses for the experimental treatment of malignancy. Information available on the heterogeneity in mechanisms of interaction between various cancer cell types, and procoagulant and fibrinolytic pathways, platelets, glycosaminoglycan-regulated growth factors and cell-adhesion molecules indicates that insightful clinical trial design may allow targeting of individual cancer cell types with agents capable of intercepting mechanisms of growth control that are relevant to specific tumor types. This paper reviews the evidence that the common anticoagulant, heparin, inhibits hepatocellular carcinoma cell proliferation and hepatocellular carcinoma tumor dissemination in experimental animals. Clinical trials of heparin performed to date have shown increased tumor response rates and survival in other tumor types. Expression of urokinase-type plasminogen activator by hepatocellular carcinoma cells enhances tumor cell proliferation, motility, invasiveness and metastatic dissemination. Inhibition of the urokinase-type plasminogen activator/plasmin system by protease inhibitors such as aprotinin (Trasylol, Bayer) have shown improvement in the clinical course of certain tumor types. These data suggest that drugs that are well-known in the field of vascular medicine may find a role in the treatment of hepatocellular carcinoma, a common tumor type that has resisted containment by other means.
The efficiency of small interfering RNA (siRNA)-induced gene knockdown is hampered by low transfection efficiency. We established a novel and simple double transfection method using specific siRNA duplexes targeted against human thrombin receptor PAR-1 in DU 145 prostate cancer cells. The initial siRNA transfection of cell suspensions followed by re-transfection of adherent cells on the following day resulted in undetectable PAR-1 mRNA and absent receptor protein. PAR-1 mRNA expression was silenced for up to five days. Functional studies showed that PAR-1 gene silencing in DU 145 cells abolished the modulating effects of thrombin on cell adhesion to the extracellular matrix proteins, fibronectin and laminin, thus demonstrating the essential role of PAR-1 in mediating thrombin effects on DU 145 cell adhesion.
The proteinase-activated receptor1 (PAR1) was characterized as a functional receptor for thrombin in cells from different tumor entities. In colon carcinoma, its function has to be defined. In this study we demonstrate that the PAR1-selective agonist peptide TFLLRN induced activation of protein kinase C isoenzymes alpha and epsilon in human HT-29 colon carcinoma cells expressing PAR1 endogeneously. On the cellular level, TFLLRN and thrombin prompted HT-29 cell migration and matrix adhesion by a PKCepsilon-dependent mechanism as concluded because of the inhibition of PAR1-mediated effects by the PKC inhibitor bisindolylmaleimide I and the PKCepsilon translocation inhibitory peptide EAVSLKPT but not by the PKC inhibitor Gö 6976. In addition, blockade of PAR1 by RWJ 56110, a selective PAR1 antagonist, fully abolished the effect of thrombin on HT-29 cell migration and adhesion. Therefore, PAR1 seems to be the responsible receptor for thrombin-induced migration and adhesion of human colon carcinoma cells including PKCepsilon as an essential signal transducer.
To explore the correlation between expression of PAR-1 and metastasis of human lung carcinoma.
Expression levels of PAR-1 were examined in surgically resected lung carcinoma specimens and corresponding lymph nodes by RT-PCR and immunohistochemistry, combined with morphometric methodology and clinicopathologic profiles.
Strong PAR-1 staining was detected in the periphery of carcinoma nests, adenocarcinomatous emboli, foci of atypical adenomatous hyperplasia adjacent to the adenocarcinoma and atypical proliferation of duct epithelium of bronchial mucous glands. The expression rates of PAR-1 were 73.8% (59/80) and 63.9% (23/36) by immunohistochemistry and RT-PCR respectively. The percentage of PAR-1 protein expression cells was significantly higher in tumors with metastasis (85.7%, 48/56) than those without (45.8%, 11/24). Morphometric study demonstrated that there were significant differences of PAR-1 protein expression levels between tumors with metastatic and those without, primary and metastatic carcinomas, primary carcinomas and benign lung tissues adjacent to the carcinoma. No significant correlation was found between PAR-1 expression level and tumor size, histological types and tumor grades. The positive rate of PAR-1 mRNA expression in the metastatic group was significantly higher than that of the non-metastatic group (78.3%, 18/23 v.s. 38.5%, 5/13).
PAR-1 expression may play an important role in determining the malignant phenotypes of lung cancers and significantly contribute to their initiation, progression and metastasis.
To determine treatment strategies and predict the clinical outcome of patients with melanoma it is important to understand the etiology of this disease. Recently, there has been some insight into molecular basis of melanoma including identification of a few of the regulatory factors and genes involved in this disease. For instance, the transcription factor AP-2 plays a tumor suppressor-like role in melanoma progression by regulating genes involved in tumor growth and metastasis. Previously, we have shown that the progression of human melanoma to the metastatic phenotype is associated with loss of AP-2 expression and deregulation of target genes such as MUC18/MCAM, c-KIT, and MMP-2. Increasing evidence demonstrates that the thrombin receptor (protease-activated receptor-1, PAR-1) plays a major role in tumor invasion and contributes to the metastatic phenotype of human melanoma. This review focuses on the role of the thrombin receptor in melanoma and its regulation by AP-2. We show that loss of AP-2 expression in metastatic melanoma cells correlates with overexpression of the thrombin receptor. Our analysis of AP-2/Sp1 complexes within the regulatory region of the thrombin receptor demonstrates that AP-2 binds the proximal 3' region of the promoter and diminishes PAR-1 expression. Levels of AP-2 and Sp1 proteins in a panel of melanoma cell lines demonstrated a marked decrease in the ratio of AP-2/Sp1, a decrease that correlated with overexpression of PAR-1 in metastatic melanoma cells. We propose that loss of AP-2 results in increased expression of the thrombin receptor, which subsequently contributes to the metastatic phenotype of melanoma by upregulating the expression of adhesion molecules, proteases, and angiogenic molecules.
Protease-activated receptors (PARs) are proposed to be involved in the invasive and metastatic processes of various types of cancer. Matrix metalloproteinase-1 (MMP-1) plays a role in cancer invasion and tissue remodelling. It has been reported that MMP-1 can alter the behavior of cancer cells through PAR-1 to promote cell migration and invasion. We considered whether the expression of PAR-1 and MMP-1 has relevance to progression in gastric cancer.
An immunohistochemical study was carried out on 129 samples of gastric cancer using anti-PAR-1 and anti-MMP-1 mouse monoclonal antibodies. Associations between immunostaining and clinicopathological factors were analyzed statistically.
There were 58 carcinomas positive for PAR-1 expression. The expression of PAR-1 was associated with the depth of wall invasion and peritoneal dissemination. There were 42 carcinomas positive for both PAR-1 and MMP-1 expression which was associated with the histological stage, depth of wall invasion, lymph node metastasis and peritoneal dissemination. These patients had a significantly poorer prognosis than those with expression-negative tumors. Multivariate analysis indicated that PAR-1 expression and combined PAR-1 and MMP-1 expression were independent prognostic factors.
The results led us to believe that the expression of PAR-1 and MMP-1 is associated with the progression of gastric cancer and an independent prognostic predictor.
The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D. L., and Chueh, S. H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 microM IP3 effects rapid release (t1/2 less than 20 s) of approximately 40% of accumulated Ca2+ releasable with 5 microM A23187. Half-maximal Ca2+ release occurs with 0.5 microM IP3, and maximal release with 3 microM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 microM IP3 rapidly releases (t1/2 less than 30 s) 10-20% of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3% polyethylene glycol (PEG). 10 microM GTP, but not guanosine 5'-(beta, gamma-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40% of releasable Ca2+) with a t1/2 value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3% PEG. Half-maximal Ca2+ release occurs with 1 microM GTP, with maximal release at 3-5 microM GTP; 20 microM GMPPNP has no effect on release and only slightly inhibits 5 microM GTP; 20 microM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 microM GTP. Using permeabilized N1E-115 cells, 5 microM GTP with 3% PEG releases greater than 50% of releasable Ca2+; without PEG, GTP still mediates approximately 30% release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.
The isolation from rabbit lung of a cofactor for thrombin-catalyzed Protein C activation is described. The lung is perfused to remove blood, homogenized, and extracted with Triton X-100. The cofactor is purified from the extract with two chromatographic columns of diisopropylphosphorothrombin-agarose. The purified cofactor is a protein with an apparent molecular weight on sodium dodecyl sulfate (SDS)-acrylamide gels of 68,000 ± 5,000 before disulfide bond reduction and 74,000 ± 6,000 after disulfide bond reduction. The co-factor activity is not inactivated permanently by SDS (1%), 8M urea, or 6M guanidinium Cl, is stable to boiling for greater than 15 min, and is stable from pH 2.0-10. Stability is determined by assay immediately after dilutions of the cofactor from the perturbant. The cofactor activity is destroyed by pepsin or β-mercaptoethanol. The stability of the cofactor in SDS and SDS and 8 M urea solutions allows correlation of the protein species visualized by protein stain with the cofactor activity determined by assay of the extracted gel slices after electrophoresis with these denaturants. The cofactor activity also co-migrates with the major protein species on alkaline disc gel electrophoresis in the presence of Lubrol PX. Neither the cofactor activity nor the protein enters the gel in the absence of Lubrol. From 10 perfused rabbit lungs, 0.7-1.5 mg of cofactor is isolated with an overall yield of 10-20%. The purified cofactor requires thrombin to activate Protein C. The rate of activation of Protein C at constant thrombin is saturable with respect to cofactor. The isolated cofactor increases the rate of Protein C activation 1000-fold. Expression of cofactor activity requires Ca2+ or other specific multivalent ions. The relationship between Ca2+ concentration and the initial rate of Protein C activation is a simple hyperbola with half-maximal stimulation at 0.2 ± 0.1 mM Ca2+. Other functional ions in order of effectiveness (in parentheses) are Tn3+ (71%), Mn2+ (52%), Sr2+ (36%), Cr2+ (23%), Co2+ (23%), Cd2+ (16%), and Gd3+ (12%). Mg2+, Ni2+, Zn2+, Cu2+, and Ba2+ were less than 5% as active as Ca2+. We conclude that the isolated cofactor has many properties in common with the other known cofactor proteins involved in the activation of vitamin K-dependent zymogens. These include a dependence on Ca2+ for function, a high affinity for the enzyme component of the complex, and an ability to increase the maximal rate of the reaction. However, the cofactor differs in that it appears to be a plasma membrane protein that does not require an intact phospholipid bilayer for biological activity.
Human α-thrombin was modified by four different procedures to identify specific active site regions required for receptor binding and stimulation of cell division. Conjugation of α-thrombin with diisopropylphosphofluoridate (DIP-F) or methylsulfonyl fluoride (MS-F) yielded catalytically inactivated preparations. Nitrations or limited proteolysis of α-thrombin led to nitro-α-thrombin or γ-thrombin preparations, respectively. Both possessed very little clotting activity but retained significant esterase activity; they were modified at regions necessary for the binding recognition of fibrinogen. Measurements of specific binding of these modified thrombins to cultured mouse, hamster, chicken, and human fibroblasts revealed no significant binding of nitro-α-thrombin or γ-thrombin. Thus, binding of α-thrombin to each of the cell types examined involved regions of α-thrombin distinct from the catalytic apparatus that are related, if not identical, to regions required for fibrinogen recognition. Binding experiments with catalytic site-conjugated DIP- or MS-α-thrombins revealed significant differences in the α-thrombin receptor among the four cell types; these thrombin forms bound as effectively as α-thrombin to mouse and hamster cells, but did not bind significantly to chick or human cells. Thus, thrombin binding to chick and human cells required the thrombin catalytic apparatus or adjacent active site regions, whereas binding to mouse or hamster cells did not. The four cell types examined all responded to the mitogenic action of α-thrombin. However, the derivative thrombin forms did not stimulate division of any of the cells significantly, with the exception of nitro-α-thrombin which possessed some residual activity for mouse cells. Since enzymatically inactive DIP- and MS-α-thrombins bound to mouse and hamster cells as effectively as active α-thrombin, the mitogenic activity of α-thrombin on these cells requires the intact catalytic apparatus of the enzyme to interact with and presumably cleave a specific protein component.
Identification of the docking interactions by which peptide agonists activate their receptors is critical for understanding signal transduction at the molecular level. The human and Xenopus thrombin receptors respond selectively to their respective hexapeptide agonists, SFLLRN and TFRIFD. A systematic analysis of human/Xenopus thrombin receptor chimeras revealed that just two human-for-Xenopus amino acid substitutions, Phe for Asn87 in the Xenopus receptor's amino-terminal exodomain and Glu for Leu260 in the second extracellular loop, conferred human receptor-like specificity to the Xenopus receptor. This observation prompted complementation studies to test the possibility that Arg5a in the human agonist peptide might normally interact with Glu260 in the human receptor. The mutant agonist peptide SFLLEN was a poor agonist at the wild type human receptor but an effective agonist at a mutant human receptor in which Glu260 was converted to Arg. An "arginine scan" of the receptor's extracellular surface revealed additional complementary mutations in the vicinity of position 260 and weak complementation at position 87 but not elsewhere in the receptor. Strikingly, a double alanine substitution that removed negative charge from the Glu260 region of the human receptor also effectively complemented the SFLLEN agonist. The functional complementation achieved with single Arg substitutions was thus due at least in part to neutralization of a negatively charged surface on the receptor and not necessarily to introduction of a new salt bridge. By contrast, charge neutralization did not account for the gain of responsiveness to SFLLRN seen in the human/Xenopus receptor chimeras. Thus two independent approaches, chimeric receptors and arginine scanning for complementary mutations, identified the Glu260 region and to a lesser degree Phe87 as important determinants of agonist specificity. These extracellular sites promote receptor responsiveness to the "correct" agonist and inhibit responsiveness to an "incorrect" agonist. They may participate directly in agonist binding or regulate agonist access to a nearby docking site.
Previous work demonstrated that alpha-thrombin promoted tumor cell adhesion to endothelium and extracellular matrix as well as enhanced the metastatic capacity of tumor cells. This study was initiated to investigate whether the thrombin effect on tumor cells is mediated through the "tethered ligand" thrombin receptor. RT-PCR analysis using primers based on the human thrombin receptors detected mRNA in human colon adenocarcinoma cells (clone A), whose authenticity was confirmed by Southern hybridization. The presence of thrombin receptor mRNA in rat (W256 carcinosarcoma) and mouse (melanoma) tumor cells was demonstrated by RT-PCR/Southern blotting using species-specific PCR primers. Sequencing of the PCR fragment of clone A cells revealed complete homology with the reported human cDNA sequence. Subsequently, tumor cells derived from three species, i.e., human, rat, and mouse, were found to express the thrombin receptor protein as revealed by immunoblotting using ligand peptide-derived mAb ATAP138, whose reactivity towards the M(r) approximately 66,000, potential thrombin receptor was blocked by preincubating the antibody with the immunogen peptide SFLLRNPNDKYEPF (TRP 14). Finally, peptides TRP 14 and TRP 7 (SFLLRNP), but not TRP 5 (FLLRN), were found to mimic alpha-thrombin in stimulating tumor cell adhesion to fibronectin, suggesting that the thrombin receptors expressed on solid tumor cells are biologically functional.
Studies in recent years have suggested that human tumor cell lines are capable of responding in vitro to hematopoietic growth factors. In the present study, we investigate the transcription of the alpha and beta subunits of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, the alpha and beta subunits of interleukin 3 (IL-3) receptor, and the single subunit of interleukin 6 (IL-6) receptor and its associated gp130 transduction protein by PCR amplification of reverse-transcribed cellular mRNA in 34 malignant cell lines derived from a variety of histological cell types. mRNA for only a single subunit polypeptide was found in a significant minority of cell lines (23%), while in 20% both the alpha and beta subunits of either the GM-CSF receptor or the IL-3 receptor were detected among a number of different histological cell types. Transcription of the gene encoding the IL-6 receptor was found in 38% of cell lines, and all lines transcribed the gp130 transduction protein, consistent with previous observations on the ubiquity of that polypeptide. In order to test the in vitro effect of exogenously added growth factors on those malignant cell lines transcribing complete cytokine receptor, either GM-CSF, IL-3, or IL-6 was added in therapeutic concentrations (20-500 ng/ml) and cellular proliferation was measured by incorporation of [3H]thymidine. No stimulation was seen at either 3 and 6 days of culture. Production of cytokine by these cell lines was investigated at the level of transcription and by assay of peptide product. None transcribed mRNA for either GM-CSF or IL-3, while 5 of 6 (STD, DOZ, ADE, Hep-2, and Detroit) expressed IL-6 mRNA. Of these latter, 2 cell lines (ADE and Hep-2) produced IL-6 as determined by bioassay, while none produced GM-CSF or IL-3 by enzyme-linked immunosorbent assay. This suggests that in the case of GM-CSF and IL-3, failure to proliferate on addition of cytokine is not due to the prior presence of endogenous production. In contrast, at least a subset of malignant cell lines may involve a closed IL-6 autocrine loop saturating cell surface sites. These findings suggest that the ability to transcribe the genes encoding cytokine receptor is by itself insufficient to render cells cytokine responsive and that malignant cells may lack the cellular machinery for cytokine-induced proliferation. This in turn suggests that therapeutic administration of either GM-CSF, IL-3, or IL-6 may involve no additional risk of tumor regrowth in vivo.
The recently cloned functional thrombin receptor is thought to be activated by thrombin cleavage of the bond between R41 and S42, followed by the insertion of the new N-terminal region ("tethered ligand") into an unknown site in the receptor. Antibodies to peptides at or near the cleavage site have been reported to inhibit thrombin-induced platelet activation to varying extents, but the precise mechanism(s) of their inhibition is unknown. We have produced: (1) a polyclonal antibody in rabbits to a peptide containing amino acids 34 to 52 (anti-TR34-52); enzyme-linked immunosorbent assays (ELISA) indicate that anti-TR34-52 contains antibodies to regions on both sides of the thrombin cleavage site; (2) two murine monoclonal antibodies (MoAbs) to a peptide containing amino acids 29 to 68; one antibody reacts primarily with residues N-terminal to the thrombin cleavage site, and the other reacts primarily with residues C-terminal to the cleavage site; and (3) a polyclonal rabbit antibody to a peptide containing amino acids 83 to 94 (anti-TR83-94). Anti-TR34-52 binds to platelets as judged by flow cytometry, and pretreating platelets with a thrombin receptor peptide ligand does not lead to loss of antibody reactivity, suggesting that platelet activation does not initiate redistribution or internalization of surface thrombin receptors. In contrast, pretreating platelets with thrombin leads to complete loss of anti-TR34-52 binding. Similarly, the binding of both MoAbs to platelets is dramatically reduced by pretreatment with thrombin. However, the binding of anti-TR83-94 is not decreased by thrombin activation, confirming that the receptor is not internalized. Anti-TR34-52 profoundly inhibits low dose thrombin-induced platelet shape change and aggregation, but the inhibition can be overcome with higher thrombin doses. However, anti-TR34-52 does not inhibit platelet aggregation induced by tethered ligand peptides. The TR34-52 peptide is a thrombin substrate, with cleavage occurring at the R41-S42 bond as judged by high performance liquid chromatography (HPLC) and platelet aggregation analysis. Anti-TR34-52 prevented cleavage of the TR34-52 peptide, suggesting that the antibody prevents platelet activation, at least in part, by preventing cleavage of the thrombin receptor. These data, although indirect, provide additional support for a thrombin activation mechanism involving thrombin cleavage of the receptor; in addition, they provide new evidence indicating that receptor cleavage is followed by loss of the N-terminal peptide, and insertion of the tethered ligand into a protected domain.
We have developed a general strategy and a versatile computer program for analysis of data from ligand-binding experiments (e.g., radioreceptor assay systems for hormones, neurotransmitters, drugs). This method provides optimal (weighted least squares) estimates of “binding parameters” (affinity constants, binding capacities, nonspecific binding) for any number of ligands reacting simultaneously with any number of receptors. This approach provides two major advantages compared with other available methods: (i): It uses an exact mathematical model of the ligand-binding system, thereby avoiding the possible biases introduced by several commonly used approximations. (ii) It uses a statistically valid, appropriately weighted least-squares curve-fitting algorithm with objective measurement of goodness of fit, thereby avoiding the subjective graphical or simplified statistical methods which may introduce bias. Additional important features include the following. (i) The level of nonspecific binding is regarded as an unknown parameter, subject to uncertainty, which must be estimated simultaneously with other parameters of the system by appropriate statistical methods. This approach provides a more accurate and precise estimate of the parameters and their standard errors. (ii) Selected parameters can be forced to share a common value, or be fixed at any desired constant value. This feature facilitates hypothesis testing by appropriate statistical methods e.g., testing whether a particular experimental manipulation results in a change in affinity (K), binding capacity (R), or both parameters. (iii) One can combine results from multiple experiments by introduction of explicit scaling or “correction” factors which compensate for the commonly observed large degree of between-experiment variation of the overall binding capacity (Bmax) while other properties of the system (e.g., K values, relative binding capacities for high- and low-affinity sites) are highly reproducible. (iv) One can characterize complex cross-reacting systems involving any number of ligands reacting simultaneously with any number of binding sites. This enables one to pool results from several curves obtained using several different ligands.
Seven different tumor cell lines (human melanoma SK MEL 28; hamster melanoma HM29; murine melanomas B16F10 and amelanotic melanoma B16a; human colon carcinoma HCT8; murine colon carcinoma CT26; and murine Lewis lung carcinoma) were treated with thrombin at 0.5-1 unit/ml and examined for their ability to bind to adherent platelets; HM29 was studied for its ability to bind to fibronectin and von Willebrand factor; CT26, B16F1, B16F10, and B16a were studied for their ability to form pulmonary metastasis after i.v. injection of thrombin-treated tumor cells; CT26 was studied for its ability to grow s.c. Five of 7 thrombin-treated tumor cell lines increased their adhesion to adherent platelets 2-to 3-fold. HM29 increased its adherence to fibronectin and von Willebrand factor 2- to 3-fold. CT26, B16F1, B16F10, and B16a increased experimental pulmonary metastasis 10- to 156-fold. Thrombin-treated CT26 cells demonstrated 2-fold greater growth in vivo after s.c. injection. The mechanism of enhanced adhesion of thrombin-treated tumor cells to platelets required the platelet integrin GPIIb-GPIIIa since it could be inhibited by agents known to block adhesion of ligands to GPIIb-GPIIIa (monoclonal antibody 10E5, tetrapeptide RGDS, disintegrin Albolabrin); as well as a "GPIIb-GPIIIa-like" structure on tumor cells since it could be inhibited by treatment of thrombin-treated tumor cells with 10E5 and RGDS. The thrombin effect on tumor cells was optimum at 1 h of incubation with thrombin, did not require active thrombin on the tumor cell surface, and did not require protein synthesis (not inhibited by cycloheximide). Thus, thrombin-treated tumor cells markedly enhance pulmonary metastasis. It is suggested that this may be secondary to thrombin-induced enhanced adhesion as well as growth of tumor cells.
We isolated a cDNA encoding a functional human thrombin receptor by direct expression cloning in Xenopus oocytes. mRNA encoding this receptor was detected in human platelets and vascular endothelial cells. The deduced amino acid sequence revealed a new member of the seven transmembrane domain receptor family with a large amino-terminal extracellular extension containing a remarkable feature. A putative thrombin cleavage site (LDPR/S) resembling the activation cleavage site in the zymogen protein C (LDPR/I) was noted 41 amino acids carboxyl to the receptor's start methionine. A peptide mimicking the new amino terminus created by cleavage at R41 was a potent agonist for both thrombin receptor activation and platelet activation. "Uncleavable" mutant thrombin receptors failed to respond to thrombin but were responsive to the new amino-terminal peptide. These data reveal a novel signaling mechanism in which thrombin cleaves its receptor's amino-terminal extension to create a new receptor amino terminus that functions as a tethered ligand and activates the receptor.
Inhibition of protein kinase C (PKC) by calmodulin is investigated and we describe the localization of inhibitory sequences within the calmodulin molecule. We present evidence that calmodulin inhibits PKC through an inhibition of the activation of PKC associated with lipid membranes: Binding of PKC to lipid vesicles is not affected, but activation is abolished. The potent calmodulin antagonist R24571 (calmidazol) did not affect the inhibition of PKC by calmodulin at concentrations up to 10(-5) M. Two tryptic fragments of calmodulin were isolated which inhibited PKC. They were only slightly less potent than intact calmodulin with an IC50 of 6 microM compared to 1 microM of intact calmodulin. They were identified as Ser38-Arg74 and His107-Lys148. Each of the inhibiting fragments contains an intact Ca2(+)-binding domain with complete helix-loop-helix structure ("EF hand"). Other calmodulin peptides showed only weak inhibitory activity. Both fragments did not stimulate cAMP phosphodiesterase even at concentrations 100-fold higher than the calmodulin concentration needed for maximal stimulation. None of the fragments acted as a calmodulin antagonist.
Protein kinase C (PKC) consists of a family of closely related enzymes highly concentrated in the CNS. These enzymes respond to the second messengers calcium (Ca2+) and diacylglycerol (DAG), to express their activities at membrane locations. Each member of this enzyme family displays distinct biochemical characteristics and is enriched in different cellular and subcellular locations. Activation of PKC in the nervous system has been implicated in the regulation of neurotransmitter release, ion channels, growth and differentiation, and neural plasticity. It is believed that an increase in the intracellular concentration of Ca2+ triggers the association of a group of PKC isozymes with the membrane where DAG interacts with PKC to stimulate the enzyme activity. Stimulation of PKC at the cellular membrane is, therefore, dependent upon the duration and magnitude of the DAG signal. The association of PKC with the membrane may also lead to a conversion of the enzyme into an effector-independent form for a sustained activation after the Ca2+ and DAG signals dissipate. Activation of PKC results in the phosphorylation of cellular proteins; however, the physiological substrates of this enzyme in the nervous system are still poorly characterized.
The Cancer and Leukemia Group B (CALGB) conducted a prospective randomized trial to evaluate the role of warfarin and alternating chemotherapy in extensive small-cell lung cancer (SCCL). After stratification for sex and performance status, patients were randomly assigned to receive chemotherapy with methotrexate, doxorubicin (Adriamycin; Adria Laboratories, Columbus, OH), cyclophosphamide, and lomustine (CCNU) (MACC), or MACC plus warfarin (MACC + W), or mitomycin, etoposide, cisplatin, and hexamethylmelamine alternating with MACC (MEPH/MACC). Warfarin was given continuously to maintain a prothrombin time of one and one half to twice the control values. A total of 328 patients were enrolled, and 294 were evaluable. There was a statistically significant advantage in objective response rates (complete [CR] and partial responses [PR], respectively) for MACC + W (17% and 50%) as compared with MACC alone (8% and 43%) or MEPH/MACC (10% and 38%) (P = .012). Both failure-free survival (P = .054 Wilcoxon test) and overall survival (P = .098 Wilcoxon test) were higher on MACC + W (median, 6.6 months and 9.3 months, respectively), as compared with MACC (5.0 months and 7.9 months) and MEPH/MACC (5.0 months and 7.9 months). Toxicity was comparable among the three arms, except for increased hemorrhagic events on MACC + W, which were life-threatening in four patients (4%), and lethal in two others (2%). These data support the role of warfarin in the treatment of SCCL, but do not establish its mechanism of action. Warfarin deserves further studies in SCCL, particularly in patients with limited disease.
Protein phosphorylation-dephosphorylation plays an important role in signal transduction in T lymphocytes. In this review, Denis Alexander and Doreen Cantrell focus on the identification, regulation and functions of the kinases and phosphatases that control phosphorylation events in T cells.
Studies of malignancy in experimental animal models have indicated that cause-effect relationships exist between coagulation activation and cancer progression. Evidence for coagulation activation in human malignancy together with favorable results from pilot clinical trials led to the establishment of a prospective, randomized therapeutic trial of warfarin in cancer. A statistically significant prolongation of survival was observed in patients with small cell carcinoma of the lung entered into this study. Demonstration of an initiator of coagulation activation together with coagulation factor intermediates and fibrin in situ associated with viable tumor cells in small cell carcinoma of the lung is consistent with the hypothesis that tumor-initiated thrombin formation might contribute to progression of this tumor type. These observations suggest novel experimental treatment strategies for small cell carcinoma of the lung. Warfarin anticoagulation may be of value in the treatment of certain other types of malignancy.
A soluble radioreceptor assay has been developed to characterize thrombin receptor activities of the human platelet membrane. 125I-Thrombin was added to platelet membranes solubilized in 1% Triton X-100, and thrombin bound to platelet receptors was separated from free thrombin by precipitation with wheat germ agglutinin (WGA) in the presence of alpha 1-acid glycoprotein as carrier. Both high affinity binding (Ki, 0.09 nM; R1, 0.30 pmol/mg protein) and moderate affinity binding (K2, 38 nM; R2, 72 pmol/mg protein) were detected in the detergent-solubilized membrane preparations and these binding parameters were in excellent agreement with values previously determined using intact platelets (Harmon, J. T., and Jamieson, G. A. (1985) Biochemistry 24, 58-64). Using the soluble radioreceptor assay, both high and moderate affinity binding was detected in highly purified preparations of glycoprotein Ib (GPIb) and glycocalicin, and the binding isotherms were identical with those of the crude detergent-solubilized membrane preparation. Treatment of detergent-solubilized membranes with increasing concentrations of a monospecific polyclonal antibody to glycocalicin resulted in the stepwise depletion of GPIb and concomitant reductions of thrombin binding activity. These results demonstrate that both high and moderate affinity binding of thrombin to platelets is completely expressed in the glycocalicin portion of GPIb.
This report identifies a component of normal human fibroblasts that forms a covalent linkage with thrombin and urokinase (urinary plasmingoen activator) and mediates most of the specific cellular binding of these proteases. This component, here named protease-nexin (PN), is both associated with the cell surface and released into the culture medium. In several ways PN resembles antithrombin III (AT3), a prominent inhibitor of thrombin in serum: PN links thrombin, probably via an ester bond; PN does not link thrombin blocked at its catalytic site serine; PN has a high-affinity heparin-binding site; and heparin greatly accelerates the rate of linkage between soluble PN and thrombin. Despite these similarities, PN and AT3 are distinct; they differ in size and are not immunologically cross-reactive. Whereas AT3 regulates the proteolytic activity of thrombin in serum, PN may regulates the activity of serine proteases at and near the cell surface.
Human alpha-thrombin is a potent chemoattractant for human monocytes, with optimum activity occurring at about 10 nanomoles
per liter. A variety of thrombins that were chemically modified to alter procoagulant or esterolytic functions showed a similar
optimum activity, but complexes of prothrombin or alpha-thrombin with either antithrombin III or hirudin did not. These findings
indicate that the regions in thrombin responsible for monocyte chemotaxis are proximate to those involved in certain protein
recognition interactions of alpha-thrombin (for example, hirudin binding) but are distinct from the catalytic site and from
certain exosites required for clotting.
Tenascin-C, a six-armed extracellular matrix glycoprotein, is expressed in a temporally and spatially restricted pattern during carcinogenesis and invasion or metastasis of carcinoma cells in association with stromal-epithelial interactions. The human epidermoid carcinoma-derived cell lines, A431 and HEp-2, which do not express tenascin-C by themselves in vitro, do express tenascin-C after transplantation into nude mice, and transforming growth factor beta 1 (TGF-beta 1) induces them to express tenascin-C in vitro. Epidermal growth factor (EGF) induced tenascin-C in these cells more effectively (about 3.5-fold greater) than did TGF-beta 1. Hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) had little effect on the induction of tenascin-C. EGF also induced other extracellular matrix components, fibronectin and laminin. Tenascin-C was also induced when the carcinoma cells were co-cultured with embryonic fibroblasts from mice which were homozygous for a null mutation in the tenascin-C gene, or when the conditioned medium from these cells was added. The induction of tenascin-C in the co-culture was reduced by treating the cells with antibodies against EGF or its receptor. The addition of EGF caused both cell types to disrupt their cytoskeleton and focal contacts as evidenced by the loss of stress fibers and vinculin plaques. EGF did neither induce tenascin-C nor affect the morphology in tenascin-C-nonproducing A549 carcinoma cells, which did not produce tenascin-C after transplantation. Thus, EGF induces tenascin-C in tenascin-C-nonproducing human carcinoma cells through EGF receptors. Furthermore, in stromalepithelial interactions, the diffusible factor EGF participates in the induction of human tenascin-C in these cells through EGF receptors.
Initial arrest of tumor cells in the microvasculature and their attachment to the endothelium and subendothelial matrix (SEM) are essential prerequisites for metastasis to occur. Factors mediating these interactions are viewed as important determinants of the tumor-cell metastatic phenotype. In this work we have studied the effects of thrombin, its analogs and its precursors on the adhesive properties and metastatic potential of tumor cells. We show that alpha-thrombin, the native form of the key coagulation enzyme, is capable of enhancing tumor-cell adhesion to both the endothelium and SEM components represented by fibronectin. Subclotting, physiological concentrations of alpha-thrombin produced a 2- to 5-fold increase in tumor-cell adhesion. A bell-shaped dose-response curve was observed, with maximal effect at 0.1 U/ml. Maximum effect occurred when cells were exposed to the agonist for 15 min and exposure for up to 4 hr resulted in enhanced tumor-cell adhesion. Prolonged incubation with thrombin resulted in a decline in the thrombin-enhanced adhesion which reached unstimulated control levels by 24 hr. Thrombin precursors and active-site-inhibited thrombin analogs only had minimal adhesion-enhancing activity; nitro- and exosite-alpha-thrombin, which retain a functional active site, mimicked, although to a lesser degree, the action of alpha-thrombin. Tumor-cell incubation with thrombin resulted in an upregulated cell-surface expression of the alpha11b beta 3 integrin, a receptor mediating interactions between tumor cells and endothelial cells, and between tumor cells and SEM. Antibodies against alpha 11b beta 3 integrin effectively inhibited thrombin-enhanced tumor-cell adhesion. Thrombin effects on tumor cells involved the PKC signal transduction pathway as thrombin-enhanced adhesion was inhibited by pre-incubation with PKC inhibitors and a transient PKC translocation from cytosol to membrane was observed following thrombin challenge. In vivo, thrombin-treated tumor cells demonstrated a 2-fold increase in their lung-colonizing ability. In contrast to the adhesion results, the metastasis-enhancing effects of alpha-thrombin were mimicked by a thrombin precursor (prothrombin) and thrombin analogs.
The thrombin receptor has been shown to be a novel member of the family of G-protein coupled receptors (Vu, T.-K. H., Hung, D.T., Wheaton, V.I., and Coughlin, S.R. (1991) Cell 64, 1057-1068). This receptor appears to be activated through a thrombin-mediated proteolytic mechanism which exposes a "tethered ligand" responsible for receptor activation. In order to investigate the initial interactions of thrombin with this receptor, we have constructed cell lines which express high levels of the human thrombin receptor and studied the binding of various forms of thrombin to the cell surface. Analysis of transfected cells with thrombin receptor monoclonal antibodies identified a particular cell line (clone #5-18) which displayed > 150,000 thrombin receptors per cell. Clone #5-18 appeared to express functional receptors since treatment with thrombin resulted in both a 15-20 fold increase of cytoplasmic phosphoinositide levels and a comparable shift in the EC50 of thrombin-mediated calcium mobilization when compared to non-transfected CHO cells. Binding of 125I-alpha-thrombin to clone #5-18 did not reach equilibrium at 37 degrees C. However, direct binding studies of 125I-alpha-, 125I-diisopropylphospho (DIP)-alpha-, and 125I-beta-thrombin to clone #5-18 demonstrated that binding at 4 degrees C was saturable and reversible for each ligand. Analysis of the binding data revealed Kd's of 0.8 nM, 0.7 nM and 9.7 nM for 125I-alpha-, 125I-DIP-alpha- and 125I-beta-thrombin respectively. Association of 125I-alpha-, DIP-alpha, and beta-thrombin could be competed by unlabelled alpha- and DIP-alpha-thrombin. Unlabelled beta-thrombin, which has a modified anion-binding exosite, was a poor competitor for 125I-alpha- and 125I-DIP-alpha-thrombin, but did compete for 125I-beta-thrombin. In addition, the hirudin54-65 peptide competed at submicromolar concentrations for the binding of alpha- and DIP-alpha-thrombin, but not for beta-thrombin. This peptide binds specifically at the anion-binding exosite of alpha-thrombin and has been shown to have a lower affinity for beta-thrombin. These results demonstrate directly a high affinity interaction between thrombin and its receptor, and suggest that an important component is the high affinity association of the thrombin receptor with the anion-binding exosite of thrombin.
Expression of the human interferon gamma receptor (IFN-gamma R) in mouse cells is not sufficient to confer biological responsiveness to human IFN-gamma and vice versa. An additional species-specific component is required for signal transduction. We identified this cofactor by expression cloning in simian COS cells stably transfected with the nonfunctional murine IFN-gamma R and a IFN-gamma-inducible reporter construct encoding the human Tac antigen (interleukin-2 receptor alpha chain, CD25). A cDNA clone was obtained that, upon stable transfection, rendered human HEp-2 cells expressing the murine IFN-gamma R fully responsive to murine IFN-gamma. This cDNA encodes a novel 332 amino acid type I transmembrane protein that belongs to the IFN receptor family and that we designate IFN-gamma R beta chain.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
active-site regions required for fibroblast receptor binding Ramakrishnan V. The anion-binding exosite is critical for and initiation of cell division– the high affinity binding of the thrombin receptor
- Bd Blackhart
- G Cuenco
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- Rm Scaborough
- Wolf
Blackhart BD, Cuenco G, Toda T, Scaborough RM, Wolf DL, active-site regions required for fibroblast receptor binding Ramakrishnan V. The anion-binding exosite is critical for and initiation of cell division. J Biol Chem 1980;255:6609– the high affinity binding of the thrombin receptor. Growth
Protease warfarin and of alternating chemotherapy in extensive nexin: a cellular component that links thrombin and plassmall-cell lung cancer by the cancer and leucemia group B. minogen activator and mediates their binding to cells
- Jb Baker
- Da Low
- Rl Simmer
- Cunningham
Baker JB, Low DA, Simmer RL, Cunningham DD. Protease warfarin and of alternating chemotherapy in extensive nexin: a cellular component that links thrombin and plassmall-cell lung cancer by the cancer and leucemia group B. minogen activator and mediates their binding to cells. Cell J Clin Oncol 1989;7:993–1002. 1980;21:37–45.
Nanagonist recognition site Immunologic analysis of the cloned
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HEp-2g cells represent a useful model for studying
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