C B Underhill

Publications of C B Underhill

• Hyaluronan synthase 3 overexpression promotes the growth of TSU prostate cancer cells.

  Authors: N Liu, F Gao, Z Han, X Xu, C B Underhill, L Zhang

  Cancer research. 08/2001; 61(13):5207-14.

  Hyaluronan synthase 3 (HAS3) is responsible for the production of both secreted and cell-associated forms of hyaluronan and is the most active of the three isoforms of this enzyme in adults. In thisHyaluronan synthase 3 (HAS3) is responsible for the production of both secreted and cell-associated forms of hyaluronan and is the most active of the three isoforms of this enzyme in adults. In this study, the cDNA for human HAS3 was cloned and characterized. The open reading frame consisted of 1659 bp coding for 553 amino acids with a deduced molecular weight of about 63,000 and isoelectric pH of 8.70. The sequence of human HAS3 displayed a 53% identity to HAS1 and a 67% identity to HAS2. It also contained a signal peptide and six potential transmembrane domains, suggesting that it was associated with the plasma membrane. To evaluate the physiological role of human HAS3, expression vectors for this protein were transfected into TSU cells (a prostate cancer cell line), and the phenotypic changes in these cells were examined. The enhanced expression of hyaluronan in the transfected cells was demonstrated by dot blot analysis and ELISA. These cells were found to differ from their vector-transfected counterparts with respect to the following: (a) they grew at a faster rate in high (but not low) density cultures; (b) conditioned media from these cells stimulated the proliferation and migration of endothelial cells; (c) when placed on the chorioallantoic membrane of chicken embryos, these cells formed large, dispersed xenografts, whereas the control transfectants formed compact masses; and (d) when injected s.c. into nude mice, the xenografts formed by HAS3 transfectants were bigger than those formed by control transfectants. Histological examination of these xenografts revealed the presence of extracellular hyaluronan that could act as conduits for the diffusion of nutrients. In addition, they had a greater number of blood vessels. However, the HAS3-transfected TSU cells did not display increased metastatic properties as judged by their ability to form lung masses after i.v. injection. These results suggested that the HAS3-induced overexpression of hyaluronan enhanced tumor cell growth, extracellular matrix deposition, and angiogenesis but was not sufficient to induce metastatic behavior in TSU cells.

• Extracellular matrix protein 1 (ECM1) has angiogenic properties and is expressed by breast tumor cells.

  Authors: Z Han, J Ni, P Smits, C B Underhill, B. Xie, Y Chen, N Liu, P Tylzanowski, D Parmelee, P Feng, I Ding, F Gao, R Gentz, D Huylebroeck, J Merregaert, L Zhang

  The FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 05/2001; 15(6):988-94.

  Tumor growth and metastasis are critically dependent on the formation of new blood vessels. The present study found that extracellular matrix protein 1 (ECM1), a newly described secretoryTumor growth and metastasis are critically dependent on the formation of new blood vessels. The present study found that extracellular matrix protein 1 (ECM1), a newly described secretory glycoprotein, promotes angiogenesis. This was initially suggested by in situ hybridization studies of mouse embryos indicating that the ECM1 message was associated with blood vessels and its expression pattern was similar to that of flk-1, a recognized marker for endothelium. More direct evidence for the role of ECM1 in angiogenesis was provided by the fact that highly purified recombinant ECM1 stimulated the proliferation of cultured endothelial cells and promoted blood vessel formation in the chorioallantoic membrane of chicken embryos. Immunohistochemical staining with specific antibodies indicated that ECM1 was expressed by the human breast cancer cell lines MDA-435 and LCC15, both of which are highly tumorigenic. In addition, staining of tissue sections from patients with breast cancer revealed that ECM1 was present in a significant proportion of primary and secondary tumors. Collectively, the results of this study suggest that ECM1 possesses angiogenic properties that may promote tumor progression.

• RGD-Tachyplesin inhibits tumor growth.

  Authors: Y Chen, X Xu, S Hong, J Chen, N Liu, C B Underhill, K Creswell, L Zhang

  Cancer research. 04/2001; 61(6):2434-8.

  Tachyplesin is an antimicrobial peptide present in leukocytes of the horseshoe crab (Tachypleus tridentatus). In this study, a synthetic tachyplesin conjugated to the integrin homing domain RGD wasTachyplesin is an antimicrobial peptide present in leukocytes of the horseshoe crab (Tachypleus tridentatus). In this study, a synthetic tachyplesin conjugated to the integrin homing domain RGD was tested for antitumor activity. The in vitro results showed that RGD-tachyplesin inhibited the proliferation of both cultured tumor and endothelial cells and reduced the colony formation of TSU prostate cancer cells. Staining with fluorescent probes of FITC-annexin V, JC-1, YO-PRO-1, and FITC-dextran indicated that RGD-tachyplesin could induce apoptosis in both tumor and endothelial cells. Western blotting showed that treatment of cells with RGD-tachyplesin could activate caspase 9, caspase 8, and caspase 3 and increase the expression of the Fas ligand, Fas-associated death domain, caspase 7, and caspase 6, suggesting that apoptotic molecules related to both mitochondrial and Fas-dependent pathways are involved in the induction of apoptosis. The in vivo studies indicated that the RGD-tachyplesin could inhibit the growth of tumors on the chorioallantoic membranes of chicken embryos and in syngenic mice.

• Metastatin: a hyaluronan-binding complex from cartilage that inhibits tumor growth.

  Authors: N Liu, R K Lapcevich, C B Underhill, Z Han, F Gao, G Swartz, S M Plum, L Zhang, S J Green

  Cancer research. 03/2001; 61(3):1022-8.

  In this study, a hyaluronan-binding complex, which we termed Metastatin, was isolated from bovine cartilage by affinity chromatography and found to have both antitumorigenic and antiangiogenicIn this study, a hyaluronan-binding complex, which we termed Metastatin, was isolated from bovine cartilage by affinity chromatography and found to have both antitumorigenic and antiangiogenic properties. Metastatin was able to block the formation of tumor nodules in the lungs of mice inoculated with B16BL6 melanoma or Lewis lung carcinoma cells. Single i.v. administration of Metastatin into chicken embryos inhibited the growth of both B16BL6 mouse melanoma and TSU human prostate cancer cells growing on the chorioallantoic membrane. The in vivo biological effect may be attributed to the antiangiogenic activity because Metastatin is able to inhibit the migration and proliferation of cultured endothelial cells as well as vascular endothelial growth factor-induced angiogenesis on the chorioallantoic membrane. In each case, the effect could be blocked by either heat denaturing the Metastatin or premixing it with hyaluronan, suggesting that its activity critically depends on its ability to bind hyaluronan on the target cells. Collectively, these results suggest that Metastatin is an effective antitumor agent that exhibits antiangiogenic activity.

• Hyaluronan and hyaluronan-binding proteins. Probes for specific detection.

  Authors: B P Toole, Q Yu, C B Underhill

  Methods in molecular biology (Clifton, N.J.). 02/2001; 171:479-85.

• Hyaluronan synthesis by epiphysial chondrocytes is regulated by growth hormone, insulin-like growth factor-1, parathyroid hormone and transforming growth factor-beta 1.

  Authors: P Pavasant, T Shizari, C B Underhill

  Matrix biology : journal of the International Society for Matrix Biology. 01/1997; 15(6):423-32.

  In a previous study, we presented evidence that the synthesis of hyaluronan by hypertrophic chondrocytes is one of the principal factors driving the interstitial expansion of the growth plateIn a previous study, we presented evidence that the synthesis of hyaluronan by hypertrophic chondrocytes is one of the principal factors driving the interstitial expansion of the growth plate (Pavasant et al., J. Cell Sci. 109: 327-334, 1996). To test this possibility further, we used two different approaches to examine the effects of hormones on the production of hyaluronan in the growth plate. In the first approach, we examined the growth plate of the lit/lit mouse that lacks growth hormone and found that its hypertrophic lacunae were smaller and contained less hyaluronan than those of wild type mice. Moreover, the ratios of hyaluronan staining density to total area of the lacunae were similar for the lit/lit and the wt/wt mice, indicating that the amount of hyaluronan is directly related to lacuna size. In the second approach, we examined the effects of hormones on segments of the epiphysial growth plate placed in organ culture. Under normal culture conditions, a band of hyaluronan staining progressed across the length of the growth plate, reflecting the maturation of chondrocytes into the hypertrophic stage. When insulin-like growth factor-1, a factor known to promote chondrocyte maturation, was added to the culture medium, the production of hyaluronan and the enlargement of the lacunae were stimulated. In contrast, when either parathyroid hormone or transforming growth factor-beta 1, both of which inhibit chondrocyte differentiation, was added to the medium of cultured segments, new pericellular hyaluronan was not detected and the lacunae did not enlarge. Taken together, these results indicate that factors that either stimulate or inhibit the maturation of epiphysial chondrocytes have a corresponding effect on the production of hyaluronan. This, in turn, further supports the importance of hyaluronan in the process of lacuna enlargement.

• Hyaluronan is exocytosed from serous, but not mucous cells, of human nasal and tracheobronchial submucosal glands.

  Authors: J N Baraniuk, T Shizari, M Sabol, M Ali, C B Underhill

  Journal of investigative medicine : the official publication of the American Federation for Clinical Research. 03/1996; 44(2):47-52.

  BACKGROUND: Hyaluronan is a large, nonsulfated glycosaminoglycan that is a major component of many extracellular matrices. Secretion of hyaluronan has been associated with inflammation in the lungsBACKGROUND: Hyaluronan is a large, nonsulfated glycosaminoglycan that is a major component of many extracellular matrices. Secretion of hyaluronan has been associated with inflammation in the lungs and other tissues. The purpose of the present study was to determine the distribution of hyaluronan in human respiratory tissues and to determine if hyaluronan was present in human nasal secretions. METHOD: Hyaluronan was localized in histological sections of human nasal and tracheobronchial mucosa using a biotinlabeled affinity-purified hyaluronan binding probe derived from cartilage proteoglycan (b-PG) (Green et al. Exp Cell Res 1988; 178:224-32). Subjects had saline nasal provocation to collect baseline nasal secretions, then ate chili peppers to provoke parasympathetic cholinergic nasal glandular secretion (gustatory provocation). Total protein and hyaluronan concentrations were measured in the lavage fluids. RESULTS: Staining for hyaluronan was intense in basement membranes of epithelium, glands and vessels, and perivascular adventitia. Many epithelial cells contained hyaluronan, but the staining intensity was less than that on the basolateral aspects of these cells. Submucosal gland serous cells contained variable amounts of hyaluronan. Hyaluronan was not present in goblet cells, submucosal gland mucous cells, or sub-basement membrane collagen deposits. The gustatory, parasympathetic stimulus induced a 4-fold increase in hyaluronan secretion (P < 0.01) and a 3.5-fold increase in total protein release (P < 0.0010). Hyaluronan accounted for 75% to 80% of the polymeric uronic acids in human nasal secretions. CONCLUSION: Hyaluronan was present human respiratory epithelial cells and submucosal gland serous cells and was exocytosed in response to parasympathetic stimulation. This distribution suggests roles in packaging cationic proteins in serous cells, cellular adhesion to basement membranes, and activation of macrophages in airway lumens.

• Hyaluronan contributes to the enlargement of hypertrophic lacunae in the growth plate.

  Authors: P Pavasant, T Shizari, C B Underhill

  Journal of cell science. 03/1996; 109 ( Pt 2):327-34.

  Histochemical staining of the epiphysial growth plate revealed that free hyaluronan (i.e. available to the staining probe) was restricted to the zone of hypertrophy, where it was located in theHistochemical staining of the epiphysial growth plate revealed that free hyaluronan (i.e. available to the staining probe) was restricted to the zone of hypertrophy, where it was located in the pericellular space between the chondrocytes and the edge of the lacunae. Furthermore, the amount of hyaluronan staining was directly proportional to the size of the lacunae. Autoradiographic analysis of growth plates cultured with isotopically labeled glucosamine indicated that at least a portion of this hyaluronan was newly synthesized by the hypertrophic chondrocytes. Since hyaluronan can adsorb large amounts of water, it is possible that it exerted a hydrostatic pressure on the surrounding territorial matrix and thereby caused the expansion of hypertrophic lacunae. To assess this possibility, segments of the growth plate were placed in organ culture under different conditions. Under normal culture conditions, a band of hyaluronan staining migrated across the segments coinciding with the enlargement of lacunae in these regions, and the segments, as a whole, increased in size. In contrast, when the segments were cultured in the presence of hyaluronidase, which degraded the pericellular hyaluronan, the lacunae did not undergo enlargement and the overall size of the segments did not increase. These results suggest that the production of hyaluronan contributes to the enlargement of hypertrophic lacunae which is important for determining both the body's stature and proportions.

• Hyaluronan in the bovine ocular anterior segment, with emphasis on the outflow pathways.

  Authors: H Gong, C B Underhill, T F Freddo

  Investigative ophthalmology & visual science. 01/1995; 35(13):4328-32.

  PURPOSE. It has been postulated that glycosaminoglycans play a role in the regulation of outflow resistance. The purpose of these studies was to localize the distribution of hyaluronan (HA) in thePURPOSE. It has been postulated that glycosaminoglycans play a role in the regulation of outflow resistance. The purpose of these studies was to localize the distribution of hyaluronan (HA) in the anterior segments of bovine eyes to understand better the possible role of HA in the outflow pathway. METHODS. Eyes from four 2-week-old calves and four 1-year-old cows were examined using a biotinylated-hyaluronan binding protein to localize HA in tissue sections of the anterior segment of bovine eyes. Various fixations and microwave irradiation were compared. The vitreous body in each section served as a positive control. Sections treated with Streptomyces hyaluronidase were used to confirm specificity. RESULTS. No significant difference in distribution of HA was found between various fixations and between calves and cows. HA was found in subconjunctival connective tissue and in intercellular spaces of limbal and conjunctival epithelium, but not in corneal epithelium. Staining was sometimes found on the surface of the corneal epithelium and endothelium, as well as on the conjunctival epithelium. No staining was found within the corneal stroma. There was HA in iris stroma, but not in the root of the iris or ciliary body stroma. HA was present in the anterior, nonfiltering part of the trabecular meshwork (TM) and surrounding collector channels and blood vessels in the sclera; to a lesser extent, it was present in the juxtacanalicular region of the TM. HA was detectable neither within trabecular beams nor filling the intertrabecular spaces. Strong staining was found, however, in the nerve bundles in the angle region. Staining for HA in vitreous was invariably positive, and in Streptomyces hyaluronidase-treated sections it was negative. CONCLUSION. HA was not uniformly distributed in the bovine TM. The distribution of HA in the flow pathway of the aqueous outflow system indicates that only a small fraction of the HA found in biochemical analyses of the bovine meshwork is located in the region where the flow resistance is thought to reside.

• Hyaluronan receptor (CD44) expression and function in human peripheral blood monocytes and alveolar macrophages.

  Authors: M Culty, T E O'Mara, C B Underhill, H Yeager, R P Swartz

  Journal of leukocyte biology. 12/1994; 56(5):605-11.

  CD44 glycoproteins are present on the surfaces of many hematopoietic cells and in some cases can bind hyaluronan, a major component of the extracellular matrix. In the present study, we have foundCD44 glycoproteins are present on the surfaces of many hematopoietic cells and in some cases can bind hyaluronan, a major component of the extracellular matrix. In the present study, we have found that newly explanted human peripheral blood monocytes (PBMs) exhibit a major CD44 band of 85 kDa, whereas autologous alveolar macrophages (AM phi) express multiple isoforms ranging from 85 to 200 kDa. Within 4 h in culture, PBMs began expressing new CD44 isoforms of 120, 150, and 180 kDa. Newly explanted AM phi specifically bound [3H]hyaluronan (135 cpm/microgram protein), but newly explanted PBMs did not. However, in vitro cultured PBM progressively acquired the ability to bind [3H]hyaluronan and exhibited specific binding of hyaluronan similar to that of AM phi (113 cpm/microgram protein) after 4 days in culture. In both case, the binding of [3H]hyaluronan was specifically inhibited by the addition of monoclonal antibody directed against CD44. AM phi readily degraded [3H]hyaluronan and reached a plateau after 4 days in culture (115 cpm/microgram protein). Newly explanted PBM exhibit no hyaluronan degradation and only a small degradative activity after 4 days in culture (6 to 11 cpm/microgram protein). Thus, CD44 expression and function appear to change as PBM mature in vitro resembling more that found in AM phi.

• Distribution of hyaluronan in the epiphysial growth plate: turnover by CD44-expressing osteoprogenitor cells.

  Authors: P Pavasant, T M Shizari, C B Underhill

  Journal of cell science. 11/1994; 107 ( Pt 10):2669-77.

  In the present study, we have examined the distribution of both hyaluronan and its receptor, CD44, during the process of endochondral ossification in the mouse tibia. Histochemical staining revealedIn the present study, we have examined the distribution of both hyaluronan and its receptor, CD44, during the process of endochondral ossification in the mouse tibia. Histochemical staining revealed that a large amount of hyaluronan was present in the lacunae located in the zone of hypertrophy, but it was greatly reduced or absent from the zone of erosion. In addition, hyaluronan was present in the cytoplasm of osteoprogenitor cells located in the zone of erosion. These cells also expressed CD44 on their surfaces, as revealed by double-label immunohistochemistry. These results suggested that the osteoprogenitor cells may use CD44 to bind and internalize hyaluronan, and subsequently degrade it with lysosomal enzymes. To test this possibility, we examined the human cell line, MG-63, which closely resembles osteoprogenitor cells. These cells produced several different forms of CD44, as determined by western blotting (85, 116 and 150 kDa). In addition, the binding of isotopically labeled hyaluronan to detergent extracts of these cells was blocked by a monoclonal antibody to CD44. Similarly, the degradation of hyaluronan by these cultured cells was also inhibited by a monoclonal antibody to CD44. To determine if these cells could remove hyaluronan from the growth plate, the cells were cultured directly on top of thin sections of the epiphysial region of long bone. After 16 hours, the sections were stained for hyaluronan. The MG-63 cells removed significant amounts of hyaluronan present in the zone of hypertrophy, and this effect was blocked by an excess of soluble hyaluronan and by a monoclonal antibody to CD44.(ABSTRACT TRUNCATED AT 250 WORDS)

• CD44 can mediate the adhesion of platelets to hyaluronan.

  Authors: I Koshiishi, M Shizari, C B Underhill

  Blood. 08/1994; 84(2):390-6.

  CD44 represents a family of glycoproteins that are present on the surfaces of some types of lymphocytes, macrophages, and epithelial cells. In the present study, we have found that CD44 is alsoCD44 represents a family of glycoproteins that are present on the surfaces of some types of lymphocytes, macrophages, and epithelial cells. In the present study, we have found that CD44 is also present on murine megakaryocytes and peripheral blood platelets as judged by immunohistochemical staining. Western blotting of platelet proteins indicated that this CD44 was predominantly of the 85-kD form. This form of CD44 also had the capacity to bind hyaluronan, because detergent extracts of platelets as well as intact platelets could bind soluble [3H]hyaluronan, and this property was blocked by antibodies directed against CD44. More importantly, isolated platelets could attach to the hyaluronan-containing extracellular matrix produced by cultured rat fibrosarcoma cells. This attachment took place in the absence of divalent cations and could be blocked by pretreating the rat fibrosarcoma cells with hyaluronidase or by the addition of an antibody to CD44. These results suggested that CD44 was responsible for the attachment of platelets to hyaluronan. Histochemical staining also showed that hyaluronan was present immediately beneath the endothelial cells of many blood vessels of various tissues, such as the dermis, lamina propria of the intestinal tract, the lungs, and the pericardium. Thus, it is possible that CD44 plays an important role in the attachment of platelets to the surface of exposed connective tissue after injury to endothelial cells.

• Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44: correlation with invasive potential.

  Authors: M Culty, M Shizari, E W Thompson, C B Underhill

  Journal of cellular physiology. 08/1994; 160(2):275-86.

  In the present study, we examined a panel of human breast cancer cell lines with regard to their expression of CD44 and ability to bind and degrade hyaluronan. The cell lines expressed varyingIn the present study, we examined a panel of human breast cancer cell lines with regard to their expression of CD44 and ability to bind and degrade hyaluronan. The cell lines expressed varying amounts of different molecular weight forms of CD44 (85-200 kDa) and, in general, those that expressed the greatest amounts of CD44 were the most invasive as judged by in vitro assays. In addition, the ability to bind and degrade hyaluronan was restricted to the cell lines expressing high levels of CD44, and both these functions were blocked by an antibody to CD44 (Hermes-1). Moreover, the rate of [3H]hyaluronan degradation was highly correlated with the amount of CD44 (r = 0.951, P < 0.0001), as well as with the invasive potential of the cells. Scatchard analysis of the [3H]hyaluronan binding of these cells revealed the existence of significant differences in both their binding capacity and their dissociation constant. To determine the source of this deviation, the different molecular weight forms of CD44 were partially separated by gel filtration chromatography. In all cell lines, the 85 kDa form was able to bind hyaluronan, although with different affinities. In contrast, not all of the high molecular weight forms of CD44 had this ability. These results illustrate the diversity of CD44 molecules in invasive tumor cells, and suggest that one of their major functions is to degrade hyaluronan.

• Hyaluronan is inversely correlated with the expression of CD44 in the dermal condensation of the embryonic hair follicle.

  Authors: C B Underhill

  The Journal of investigative dermatology. 01/1994; 101(6):820-6.

  Previously, we have shown that CD44 (the hyaluronan receptor) was involved in the degradation of hyaluronan. In the present study, we examined the distribution of CD44 and hyaluronan in the skin ofPreviously, we have shown that CD44 (the hyaluronan receptor) was involved in the degradation of hyaluronan. In the present study, we examined the distribution of CD44 and hyaluronan in the skin of embryonic and mature mice. During embryonic development, CD44 was prominently expressed by the condensed mesenchymal cells involved in the formation of the hair follicles, but was absent from the surrounding interstitial cells. The cells of the dermal condensation expressed CD44 throughout the development of the hair follicle; however, once the hair follicle reached maturity, the mesenchymal cells of the dermal papilla no longer expressed this molecule. In contrast to the above, the distribution of hyaluronan was reversed from that of CD44. Hyaluronan was widespread throughout the embryonic dermis, but was conspicuously absent from the regions of the dermal condensation. This arrangement persisted through the development of the hair follicle; however, in the mature hair follicle, hyaluronan reappeared in the dermal papilla. Thus, in the embryonic dermis, the expression of CD44 and hyaluronan were complementary to each other. However, in the adult skin, only minor changes were detected in the levels of CD44 and hyaluronan associated with the cells of the dermal condensation during the hair cycle. When organ cultures of embryonic mouse skin were treated with Streptomyces hyaluronidase, the interstitial mesenchymal cells became compacted, indicating that the removal of hyaluronan leads to the condensation of these cells. The results of this study are consistent with the hypothesis that the expression of CD44 by the inductive mesenchymal cells allows them to degrade hyaluronan in a localized region, leading to formation and maintenance of the dermal condensation.

• CD44 positive macrophages take up hyaluronan during lung development.

  Authors: C B Underhill, H A Nguyen, M Shizari, M Culty

  Developmental biology. 03/1993; 155(2):324-36.

  In the present study, we examined the expression and distribution of both hyaluronan and its cell-surface receptor (CD44) during lung development in the mouse. Hyaluronan was detected by a specificIn the present study, we examined the expression and distribution of both hyaluronan and its cell-surface receptor (CD44) during lung development in the mouse. Hyaluronan was detected by a specific binding probe, termed b-PG, which is a biotinylated mixture of proteoglycan and link protein from cartilage. Using this probe in an enzyme-linked assay, the amount of hyaluronan in relation to protein content was found to decrease as lung development progressed. In addition, histochemical staining of the embryonic lungs revealed that during early stages, relatively large amounts of hyaluronan were present in the interstitium. However, as development progressed, much of this was lost, and in the adult, hyaluronan was restricted to the regions surrounding the major blood vessels, bronchi, and bronchioles. In contrast to hyaluronan, the amount of CD44 increased as a function of development, as determined by the rat monoclonal antibody, KM-201. Histochemical staining with this antibody showed that the receptor was primarily associated with macrophages and to a lesser extent with adult bronchial and bronchiolar epithelium, vascular smooth muscle, and endothelial cells. As development progressed, the macrophages expressing CD44 increased in number, and this increase was temporarily correlated with the decrease in hyaluronan content. In addition, histochemical staining revealed that some of these macrophages contained hyaluronan in their cytoplasm, suggesting that macrophages had internalized hyaluronan from the extracellular matrix. This possibility was further supported by the fact that when newborn mice were injected with the KM-201 monoclonal antibody, which blocks the interaction between hyaluronan and the receptor, the number of hyaluronan-containing macrophages in the lungs decreased while the concentration of hyaluronan increased. Taken together, these results suggest that macrophages can internalize hyaluronan during lung development and could possibly play a significant role in its removal.

• A novel biotinylated probe specific for hyaluronate. Its diagnostic value in diffuse malignant mesothelioma.

  Authors: N Azumi, C B Underhill, E Kagan, K Sheibani

  The American journal of surgical pathology. 03/1992; 16(2):116-21.

  Diffuse malignant mesotheliomas are known to secrete a large amount of hyaluronate, whereas adenocarcinomas produce predominantly neutral mucins. In the present study, we assessed the diagnosticDiffuse malignant mesotheliomas are known to secrete a large amount of hyaluronate, whereas adenocarcinomas produce predominantly neutral mucins. In the present study, we assessed the diagnostic usefulness of a new, highly specific and sensitive hyaluronate binding probe to discriminate between mesotheliomas and adenocarcinomas. We studied 33 mesotheliomas and 37 adenocarcinomas in order to establish specific diagnostic criteria for using the hyaluronate binding probe. Of the adenocarcinomas, only three showed significant positive staining for hyaluronate (8%). By contrast, all the mesotheliomas exhibited positive staining for hyaluronate. Furthermore, the staining reaction was classed as moderate or greater in 26 mesotheliomas (79%), thus suggesting the utility of this probe in the differential diagnosis of malignant mesothelioma versus adenocarcinoma. We conclude that strong cytoplasmic or membranous staining for hyaluronate is highly predictive of malignant mesothelioma. The hyaluronate binding probe should therefore be considered an important adjunct to be used in combination with electron microscopy and immunohistochemistry in the histologic diagnosis of diffuse malignant mesothelioma.

• The hyaluronan receptor (CD44) participates in the uptake and degradation of hyaluronan.

  Authors: M Culty, H A Nguyen, C B Underhill

  The Journal of cell biology. 03/1992; 116(4):1055-62.

  The hyaluronan receptor belongs to the polymorphic family of CD44 glycoproteins, which have been implicated in a variety of cellular functions including adhesion to hyaluronan and collagen, theThe hyaluronan receptor belongs to the polymorphic family of CD44 glycoproteins, which have been implicated in a variety of cellular functions including adhesion to hyaluronan and collagen, the binding of lymphocytes to high endothelial cells during extravasation, and conferring metastatic potential to carcinoma cells. Here, we demonstrate that the receptor also participates in the uptake and degradation of hyaluronan by both transformed fibroblasts (SV-3T3 cells) and alveolar macrophages. These cells were incubated with isotopically labeled hyaluronan for various periods of time, and the extent of degradation was determined by either molecular-sieve chromatography or centrifugation through Centricon 30 microconcentrators. The macrophages degraded the hyaluronan at a faster rate than the SV-3T3 cells, which may reflect the fact that they contained a greater number of receptors. More importantly, in both cell types, the degradation of hyaluronan was specifically blocked by antibodies directed against the receptor. However, the receptor by itself did not have the ability to degrade hyaluronan, since preparations of SV-3T3 membranes containing the receptor did not break down hyaluronan. Subsequent experiments revealed that macrophages can internalize fluorescein-tagged hyaluronan, and this process was blocked by antibodies against the receptor. Furthermore, the subsequent degradation of hyaluronan was inhibited by agents that block the acidification of lysosomes (chloroquine and NH4Cl). Thus, the most likely explanation for these results is that the receptor mediates the uptake of hyaluronan into the cell where it can be degraded by acid hydrolases in lysosomes. The ability of cells expressing the receptor to degrade hyaluronan may be important during tissue morphogenesis and cell migration.

• The hyaluronate receptor is preferentially expressed on proliferating epithelial cells.

  Authors: A M Alho, C B Underhill

  The Journal of cell biology. 05/1989; 108(4):1557-65.

  In the present study, we have examined the distribution of the hyaluronate receptor as well as hyaluronate itself in a variety of adult tissues. The hyaluronate receptor was localized with aIn the present study, we have examined the distribution of the hyaluronate receptor as well as hyaluronate itself in a variety of adult tissues. The hyaluronate receptor was localized with a monoclonal antibody, termed K-3, while hyaluronate was localized using proteolytic fragments of cartilage proteoglycan. Staining with the K-3 monoclonal antibody revealed that the hyaluronate receptor was present in a variety of epithelia including the skin, cheek, tongue, esophagus, vagina, intestines, oviduct, and bladder. However, it was notably absent from epithelial cells of the cornea and stomach as well as from endothelial cells of blood vessels. When present, the hyaluronate receptor was preferentially located in regions of active cell growth, such as in the basal layers of stratified epithelium and at the base of the crypts of Lieberkuhn in intestinal epithelium. A similar phenomenon was observed in cultured 3T3 cells. Cultures of 3T3 cells that were actively proliferating were found to have greater amounts of the receptor than their nonproliferating counterparts. When the various tissues were examined for hyaluronate, it was found to have a widespread distribution, being present in most of the basement membranes and between the cells in stratified epithelium. Indeed, in many cases, the distribution of hyaluronate closely paralleled that of the hyaluronate receptor. These results suggest that the interaction between hyaluronate and its receptor is involved in cell-to-substratum adhesion.

• The interaction of hyaluronate with the cell surface: the hyaluronate receptor and the core protein.

  Authors: C B Underhill

  Ciba Foundation symposium. 02/1989; 143:87-99; discussion 100-6, 281-5.

  Two distinct mechanisms are discussed by which hyaluronate interacts with the surfaces of cells: first, through a receptor which binds to hyaluronate with high affinity; and second, through aTwo distinct mechanisms are discussed by which hyaluronate interacts with the surfaces of cells: first, through a receptor which binds to hyaluronate with high affinity; and second, through a hydrophobic protein which is covalently linked to hyaluronate. The hyaluronate receptor is a transmembrane glycoprotein of Mr 85,000 which appears to interact with actin filaments of the cytoskeleton. It recognizes a sequence of six sugar residues of hyaluronate and also binds to chondroitin sulphate with a lower affinity. On the cell surface the receptors bind hyaluronate in cooperative fashion whereby two or more receptors can bind to the same molecule of hyaluronate, resulting in a high affinity. Immunohistochemical staining with a monoclonal antibody to the receptor indicates that it is present on epithelia, macrophages and other mononuclear phagocytes as well as some type of neurons. In epithelia the receptors presumably help to mediate cell attachment to the basement membrane which is often rich in hyaluronate. The receptor also appears to be preferentially expressed on proliferating epithelial cells and may serve as a marker for some types of carcinomas. Macrophages and related cells also have large amounts of the receptor, where it may serve in cell migration and/or in the homing of the cells to certain types of tissues. Recent studies have suggested that cell surface hyaluronate is covalently attached to a membrane-associated core protein. First, if cultured rat fibrosarcoma cells are fixed with glutaraldehyde the cell surface hyaluronate remains associated with the cells even under conditions expected to break non-covalent bonds. Second, when cell surface hyaluronate is partitioned with Triton X-114 a significant fraction is recovered in the hydrophobic phase, suggesting attachment to a hydrophobic protein. And finally, the binding of cell surface hyaluronate to nitrocellulose appears to be mediated through a covalent linkage to a protein.

• Aggregation of macrophages and fibroblasts is inhibited by a monoclonal antibody to the hyaluronate receptor.

  Authors: S J Green, G Tarone, C B Underhill

  Experimental cell research. 11/1988; 178(2):224-32.

  To examine the role of the hyaluronate receptor in cell to cell adhesion, we have employed the K-3 monoclonal antibody (MAb) which specifically binds to the hyaluronate receptor and blocks itsTo examine the role of the hyaluronate receptor in cell to cell adhesion, we have employed the K-3 monoclonal antibody (MAb) which specifically binds to the hyaluronate receptor and blocks its ability to interact with hyaluronate. In the first set of experiments, we investigated the spontaneous aggregation of SV-3T3 cells, which involves two distinct mechanisms, one of which is dependent upon the presence of divalent cation and the other is independent. The divalent cation-independent aggregation was found to be completely inhibited by both intact and Fab fragments of the K-3 MAb. In contrast, the K-3 MAb had no effect on the divalent cation-dependent aggregation of cells. In a second set of experiments, we examined alveolar macrophages. The presence of hyaluronate receptors on alveolar macrophages was demonstrated by the fact that detergent extracts of these cells could bind [3H]hyaluronate, and this binding was blocked by the K-3 MAb. Immunoblot analysis of alveolar macrophages showed that the hyaluronate receptor had a Mr of 99,500, which is considerably larger than the 85,000 Mr for that on BHK cells. When hyaluronate was added to suspensions of alveolar macrophages, the cells were induced to aggregate. This effect was inhibited by the K-3 MAb, suggesting that the hyaluronate-induced aggregation was mediated by the receptor.