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ABSTRACT: In addition to the well-known regulating effects of leptin on energy balance and glucose homeostasis through the central nervous system, circulating leptin has a direct effect on pancreatic islet and insulin secretion through its receptor (OBRb). The LIM-homeodomain transcription factor Isl-1 is expressed in all classes of pancreatic endocrine cells, and is involved in regulating both islet development and insulin secretion. Both OBRb and Isl-1 mutations result in obesity-related diabetes. However, the interactions and physiological significance of leptin and Isl-1 in pancreatic islets remain to be established. Here, we show that all leptin target cells in pancreatic islets and NIT beta cell express Isl-1. Both in vivo and in vitro results demonstrate that leptin suppresses Isl-1 expression and insulin secretion in islet in physiological and patho-physiological condition e.g., high fat diet (HFD). This effect of leptin on insulin secretion is lost in leptin receptor-defective db/db and Isl-1-inducible knockout (IKO) mice. We conclude that the action of leptin on insulin secretion is, at least partly, mediated by Isl-1. Another new finding of the present study is that Isl-1 acts as a direct downstream target of leptin signaling molecule STAT3 to influence the effect of leptin on insulin secretion. While inversely, insulin has feedback regulating effects on Isl-1 expression through JAK-STAT3 pathway. These findings are crucial for understanding the mechanisms regulating insulin secretion and metabolism in related diseases, such as obesity and type 2 diabetes.
Journal of Biological Chemistry 03/2013; · 4.77 Impact Factor
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ABSTRACT: Enterovirus 71 (EV71) is the major causative pathogen of hand, foot, and mouth disease (HFMD). Its pathogenicity is not fully understood, but innate immune evasion is likely a key factor. Strategies to circumvent the initiation and effector phases of anti-viral innate immunity are well known; less well known is whether EV71 evades the signal transduction phase regulated by a sophisticated interplay of cellular and viral proteins. Here, we show that EV71 inhibits anti-viral type I interferon (IFN) responses by targeting the mitochondrial anti-viral signaling (MAVS) protein-a unique adaptor molecule activated upon retinoic acid induced gene-I (RIG-I) and melanoma differentiation associated gene (MDA-5) viral recognition receptor signaling-upstream of type I interferon production. MAVS was cleaved and released from mitochondria during EV71 infection. An in vitro cleavage assay demonstrated that the viral 2A protease (2A(pro)), but not the mutant 2A(pro) (2A(pro)-110) containing an inactivated catalytic site, cleaved MAVS. The Protease-Glo assay revealed that MAVS was cleaved at 3 residues between the proline-rich and transmembrane domains, and the resulting fragmentation effectively inactivated downstream signaling. In addition to MAVS cleavage, we found that EV71 infection also induced morphologic and functional changes to the mitochondria. The EV71 structural protein VP1 was detected on purified mitochondria, suggesting not only a novel role for mitochondria in the EV71 replication cycle but also an explanation of how EV71-derived 2A(pro) could approach MAVS. Taken together, our findings reveal a novel strategy employed by EV71 to escape host anti-viral innate immunity that complements the known EV71-mediated immune-evasion mechanisms.
PLoS Pathogens 03/2013; 9(3):e1003231. · 9.13 Impact Factor
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ABSTRACT: Pro-opiomelanocortin (POMC) is a common precursor of melanocortin-related peptides in the pituitary and primarily regulated by CRF. Our results show that miR-375 is highly expressed in the mouse pituitary gland and located specifically in the intermediate lobe of pituitary. The functional studies show that the forced inhibition of endogenous miR-375 in AtT-20 mouse pituitary tumor cells and in the intermediate lobe of the pituitary gland significantly increases POMC expression, while miR-375 over-expression down- regulates POMC expression and ACTH secretion stimulated by corticotropin- releasing factor (CRF). This function of miR-375 is accomplished by its binding to the 3'-untranslated region (3'UTR) of mitogen activated protein kinase kinase kinase-8 (MAP3K8). Our results here have demonstrated that miR-375 acts as a negative regulating molecule mediating the signaling pathway of CRF and affecting POMC expression by targeting MAP3K8, which subsequently down-regulates ERK1/2 phosphorylation and NGFI-B transcription activity. Taken together, our results show that miR-375 is a novel negative regulator of POMC expression and related hormone secretion.
Journal of Biological Chemistry 02/2013; · 4.77 Impact Factor
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ABSTRACT: Although microRNAs (miRNAs) have been implicated in fine-tuning gene networks, the roles of mmu-mir-143 (miR-143) in mammalian ovary development have not been studied in vitro. We investigated the expression and function of miR-143 in the mouse ovary during primordial follicle formation. Real-time polymerase chain reaction analysis showed that miR-143 expression increased during primordial follicle formation from 15.5 days post-coitus to 4 days post-partum. miR-143 was located in pregranulosa cells by in situ hybridization. To study the function of miR-143 in primordial follicle formation we established an electroporation transfection model in vitro that allowed miR-143 expression to be efficiently upregulated and inhibited in cultured ovaries. Further studies showed that miR-143 inhibited the formation of primordial follicles by suppressing pregranulosa cell proliferation and downregulating the expression of genes related to the cell cycle. These findings suggest that miR-143 is critical for the formation of primordial follicles and regulates ovarian development and function.
Frontiers in Bioscience 01/2013; 18:588-97. · 3.52 Impact Factor
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ABSTRACT: Peptides derived from the C-terminal heptad repeat (C peptides) of HIV-1 gp41 are potent inhibitors against virus entry. However, development of a short C peptide possessing high anti-HIV potency is considered a daunting challenge. We recently discovered that the residues Met626 and Thr627 preceding the pocket-binding domain of the C peptide adopt a unique M-T hook structure that is crucial for the design of HIV-1 fusion inhibitors. In this study, we first presented a proof-of-concept prototype that the M-T hook residues can dramatically improve the antiviral activity and thermostability of a short C peptide. We then generated a 24-mer peptide termed MT-SC22EK by incorporating the M-T hook structure to the N terminus of the poorly active short C peptide SC22EK. Amazingly, MT-SC22EK inhibited HIV-1-mediated cell fusion and infection at a level comparable to C34, T1249, SC29EK, and sifuvirtide, and it was highly active against diverse HIV-1 subtypes and variants, including those T20 (enfuvirtide) and SC29EK-resistant viruses. The high-resolution crystal structure of MT-SC22EK reveals the N-terminal M-T hook conformation folded by incorporated Met626 and Thr627 and identifies the C-terminal boundary critical for the anti-HIV activity. Collectively, our studies provide new insights into the mechanisms of HIV-1 fusion and its inhibition.
The FASEB Journal 12/2012; · 5.71 Impact Factor
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ABSTRACT: CP621-652 is a potent HIV-1 fusion inhibitor peptide derived from the C-terminal heptad repeat of gp41. We recently identified that its N-terminal residues Met-626 and Thr-627 adopt a unique hook-like structure (termed M-T hook) thus stabilizing the interaction of the inhibitor with the deep pocket on the N-terminal heptad repeat. In this study, we further demonstrated that the M-T hook structure is a key determinant of CP621-652 in terms of its thermostability and anti-HIV activity. To directly define the structure and function of the M-T hook, we generated the peptide MT-C34 by incorporating Met-626 and Thr-627 into the N terminus of the C-terminal heptad repeat-derived peptide C34. The high resolution crystal structure (1.9 Å) of MT-C34 complexed by an N-terminal heptad repeat-derived peptide reveals that the M-T hook conformation is well preserved at the N-terminal extreme of the inhibitor. Strikingly, addition of two hook residues could dramatically enhance the binding affinity and thermostability of 6-helix bundle core. Compared with C34, MT-C34 exhibited significantly increased activity to inhibit HIV-1 envelope-mediated cell fusion (6.6-fold), virus entry (4.5-fold), and replication (6-fold). Mechanistically, MT-C34 had a 10.5-fold higher increase than C34 in blocking 6-helix bundle formation. We further showed that MT-C34 possessed higher potency against T20 (Enfuvirtide, Fuzeon)-resistant HIV-1 variants. Therefore, this study provides convincing data for our proposed concept that the M-T hook structure is critical for designing HIV-1 fusion inhibitors.
Journal of Biological Chemistry 08/2012; 287(41):34558-68. · 4.77 Impact Factor
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ABSTRACT: CP32M is a newly designed peptide fusion inhibitor possessing potent anti-HIV activity, especially against T20-resistant HIV-1 strains. In this study, we show that CP32M can efficiently inhibit a large panel of diverse HIV-1 variants, including subtype B', CRF07_BC, and CRF01_AE recombinants and naturally occurring or induced T20-resistant viruses. To elucidate its mechanism of action, we determined the crystal structure of CP32M complexed with its target sequence. Differing from its parental peptide, CP621-652, the (621)VEWNEMT(627) motif of CP32M folds into two α-helix turns at the N terminus of the pocket-binding domain, forming a novel layer in the six-helix bundle structure. Prominently, the residue Asn-624 of the (621)VEWNEMT(627) motif is engaged in the polar interaction with a hydrophilic ridge that borders the hydrophobic pocket on the N-terminal coiled coil. The original inhibitor design of CP32M provides several intra- and salt bridge/hydrogen bond interactions favoring the stability of the helical conformation of CP32M and its interactions with N-terminal heptad repeat (NHR) targets. We identified a novel salt bridge between Arg-557 on the NHR and Glu-648 of CP32M that is critical for the binding of CP32M and resistance against the inhibitor. Therefore, our data present important information for developing novel HIV-1 fusion inhibitors for clinical use.
Journal of Biological Chemistry 06/2012; 287(32):26618-29. · 4.77 Impact Factor
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ABSTRACT: The core structure of HIV-1 gp41 is a stable six-helix bundle (6-HB) folded by its trimeric N- and C-terminal heptad repeats (NHR and CHR). We previously identified that the (621)QIWNNMT(627) motif located at the upstream region of gp41 CHR plays critical roles for the stabilization of the 6-HB core and peptide CP621-652 containing this motif is a potent HIV-1 fusion inhibitor, however, the molecular determinants underlying the stability and anti-HIV activity remained elusive. In this study, we determined the high-resolution crystal structure of CP621-652 complexed by T21. We find that the (621)QIWNNMT(627) motif does not maintain the α-helical conformation. Instead, residues Met(626) and Thr(627) form a unique hook-like structure (denoted as M-T hook), in which Thr(627) redirects the peptide chain to position Met(626) above the left side of the hydrophobic pocket on the NHR trimer. The side chain of Met(626) caps the hydrophobic pocket, stabilizing the interaction between the pocket and the pocket-binding domain. Our mutagenesis studies demonstrate that mutations of the M-T hook residues could completely abolish HIV-1 Env-mediated cell fusion and virus entry, and significantly destabilize the interaction of NHR and CHR peptides and reduce the anti-HIV activity of CP621-652. Our results identify an unusual structural feature that stabilizes the six-helix bundle, providing novel insights into the mechanisms of HIV-1 fusion and inhibition.
Journal of Biological Chemistry 04/2012; 287(24):20281-9. · 4.77 Impact Factor
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ABSTRACT: Estrogens and androgens play important roles in regulating the hormone-secreting functions of the pituitary gland by binding to their corresponding receptors. However, the expression of estrogen receptors (ERs) and the androgen receptor (AR) and the cell types containing ERs and AR in the anterior pituitary gland of adult chickens have not been well-studied. In this study, the distribution of ERα, AR and their corresponding cell types in the anterior pituitary gland of adult cockerels was detected by immunohistochemistry. The results showed that ERα was expressed in 68.63 % of luteinizing hormone (LH) producing cells but was not found in thyrotropes, lactotropes, somatotropes, corticotropes and folliculo-stellate (FS) cells. Pituitary hormone and AR double labeling results showed that about 37 % of LH cells and 50 % of thyroid-stimulating hormone (TSH) producing cells expressed AR, respectively. In contrast, less than 1 % of the somatotropes had an AR positive signal and AR signals were not detected in lactotropes, corticotropes or FS cells. In addition, there were only a few AR and ERα dual-labeled cells observed. These novel results provide evidence for a cell-specific distribution of ERα and AR in the anterior pituitary from adult cockerels by immunohistochemistry. The different distributions of ERα and AR in the LH cells suggest that the feedback-regulating mechanisms of estrogen and androgen on the pituitary hormones secretion are different. The functions and related mechanisms still need to be elucidated further.
Cell and Tissue Research 03/2012; 348(3):551-8. · 3.11 Impact Factor
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ABSTRACT: Sifuvirtide (SFT) is an electrostatically constrained α-helical peptide fusion inhibitor showing potent anti-HIV activity, good safety, and pharmacokinetic profiles, and it is currently under phase II clinical trials in China. In this study, we demonstrate its potent and broad anti-HIV activity by using diverse HIV-1 subtypes and variants, including subtypes A, B, and C that dominate the AIDS epidemic worldwide, and subtypes B', CRF07_BC, and CRF01_AE recombinants that are currently circulating in China, and those possessing cross-resistance to the first and second generation fusion inhibitors. To elucidate its mechanism of action, we determined the crystal structure of SFT in complex with its target N-terminal heptad repeat region (NHR) peptide (N36), which fully supports our rational inhibitor design and reveals its key motifs and residues responsible for the stability and anti-HIV activity. As anticipated, SFT adopts fully helical conformation stabilized by the multiple engineered salt bridges. The designing of SFT also provide novel inter-helical salt bridges and hydrogen bonds that improve the affinity of SFT to NHR trimer. The extra serine residue and acetyl group stabilize α-helicity of the N-terminal portion of SFT, whereas Thr-119 serves to stabilize the hydrophobic NHR pocket. In addition, our structure demonstrates that the residues critical for drug resistance, located at positions 37, 38, 41, and 43 of NHR, are irreplaceable for maintaining the stable fusogenic six-helix bundle structure. Our data present important information for developing SFT for clinical use and for designing novel HIV fusion inhibitors.
Journal of Biological Chemistry 02/2012; 287(9):6788-96. · 4.77 Impact Factor
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ABSTRACT: Sifuvirtide (SFT) is an electrostatically constrained α-helical peptide fusion inhibitor showing potent anti-HIV activity,
good safety, and pharmacokinetic profiles, and it is currently under phase II clinical trials in China. In this study, we
demonstrate its potent and broad anti-HIV activity by using diverse HIV-1 subtypes and variants, including subtypes A, B,
and C that dominate the AIDS epidemic worldwide, and subtypes B′, CRF07_BC, and CRF01_AE recombinants that are currently circulating
in China, and those possessing cross-resistance to the first and second generation fusion inhibitors. To elucidate its mechanism
of action, we determined the crystal structure of SFT in complex with its target N-terminal heptad repeat region (NHR) peptide
(N36), which fully supports our rational inhibitor design and reveals its key motifs and residues responsible for the stability
and anti-HIV activity. As anticipated, SFT adopts fully helical conformation stabilized by the multiple engineered salt bridges.
The designing of SFT also provide novel inter-helical salt bridges and hydrogen bonds that improve the affinity of SFT to
NHR trimer. The extra serine residue and acetyl group stabilize α-helicity of the N-terminal portion of SFT, whereas Thr-119
serves to stabilize the hydrophobic NHR pocket. In addition, our structure demonstrates that the residues critical for drug
resistance, located at positions 37, 38, 41, and 43 of NHR, are irreplaceable for maintaining the stable fusogenic six-helix
bundle structure. Our data present important information for developing SFT for clinical use and for designing novel HIV fusion
inhibitors.
Journal of Biological Chemistry 02/2012; 287(9):6788-6796. · 4.77 Impact Factor
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ABSTRACT: In the competition for niches in natural resources, Pseudomonas aeruginosa utilizes the type VI secretion system to inject the toxic protein effector Tse2 into bacteria on cell-cell contact. The cytoplasm toxin immunity protein Tsi2 can neutralize Tse2 by physical interaction with the toxin, providing essential protection from toxin activity. Except for orthologues in P. aeruginosa, Tsi2 antitoxin does not share detectable sequence homology with known proteins in public databases. The mechanism underlying toxin neutralization by Tsi2 remains unknown. We report here the crystal structure of Tsi2 at 2.28 Å resolution. Our structural and biophysical analyses demonstrate that the antitoxin adopts a previously unobserved superhelical conformation. Tsi2 is highly thermostable in the absence of the toxin in solution. Tsi2 assembles a dimer with 2-fold rotational symmetry, similar to that observed in other toxin-antitoxin systems. Dimerization is essential for the stable folding of Tsi2.
Journal of Molecular Biology 01/2012; 417(4):351-61. · 4.00 Impact Factor
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ABSTRACT: Albuvirtide (ABT) is a 3-maleimimidopropionic acid (MPA)-modified peptide HIV fusion inhibitor that can irreversibly conjugate to serum albumin. Previous studies demonstrated its in vivo long half-life and potent anti-HIV activity. Here, we focused to characterize its biophysical properties and evaluate its antiviral spectrum. In contrast to T20 (Enfuvirtide, Fuzeon), ABT was able to form a stable α-helical conformation with the target sequence and block the fusion-active six-helix bundle (6-HB) formation in a dominant-negative manner. It efficiently inhibited HIV-1 Env-mediated cell membrane fusion and virus entry. A large panel of 42 HIV-1 pseudoviruses with different genotypes were constructed and used for the antiviral evaluation. The results showed that ABT had potent inhibitory activity against the subtypes A, B and C that predominate the worldwide AIDS epidemics, and subtype B', CRF07_BC and CRF01_AE recombinants that are currently circulating in China. Furthermore, ABT was also highly effective against HIV-1 variants resistant to T20. Taken together, our data indicate that the chemically modified peptide ABT can serve as an ideal HIV-1 fusion inhibitor.
PLoS ONE 01/2012; 7(3):e32599. · 4.09 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) play vital regulatory roles in many cellular processes. The expression of miRNA (miR)-34c is highly enriched in adult mouse testis, but its roles and underlying mechanisms of action are not well understood.
In the present study, we show that miR-34c is detected in mouse pachytene spermatocytes and continues to be highly expressed in spermatids. To explore the specific functions of miR-34c, we have established an in vivo model by transfecting miR-34c inhibitors into primary spermatocytes to study the loss-of-function of miR-34c. The results show that silencing of miR-34c significantly increases the Bcl-2/Bax ratio and prevents germ cell from apoptosis induced by deprivation of testosterone. Moreover, ectopic expression of the miR-34c in GC-2 cell trigger the cell apoptosis with a decreased Bcl-2/Bax ratio and miR-34c inhibition lead to a low spontaneous apoptotic ratio and an increased Bcl-2/Bax ratio. Furthermore, ectopic expression of miR-34c reduces ATF1 protein expression without affecting ATF1 mRNA level via directly binding to ATF1's 3'UTR, indicating that ATF1 is one of miR-34c's target genes. Meanwhile, the knockdown of ATF1 significantly decreases the Bcl-2/Bax ratio and triggers GC-2 cell apoptosis. Inhibition of miR-34c does not decrease the GC-2 cell apoptosis ratio in ATF1 knockdown cells.
Our study shows for the first time that miR-34c functions, at least partially, by targeting the ATF1 gene in germ cell apoptosis, providing a novel mechanism with involvement of miRNA in the regulation of germ cell apoptosis.
PLoS ONE 01/2012; 7(3):e33861. · 4.09 Impact Factor
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ABSTRACT: EV71 is the primary pathogenic cause of hand-foot-mouth disease (HFMD), but an effective antiviral drug currently is unavailable. Rupintrivir, an inhibitor against human rhinovirus (HRV), has potent antiviral activities against EV71. We determined the high-resolution crystal structures of the EV71 3C(pro)/rupintrivir complex, showing that although rupintrivir interacts with EV71 3C(pro) similarly to HRV 3C(pro), the C terminus of the inhibitor cannot accommodate the leaving-group pockets of EV71 3C(pro). Our structures reveal that EV71 3C(pro) possesses a surface-recessive S2' pocket that is not present in HRV 3C(pro) that contributes to the additional substrate binding affinity. Combined with mutagenic studies, we demonstrated that catalytic Glu71 is irreplaceable for maintaining the overall architecture of the active site and, most importantly, the productive conformation of catalytic His40. We discovered the role of a previously uncharacterized residue, Arg39 of EV71 3C(pro), that can neutralize the negative charge of Glu71, which may subsequently assist deprotonation of His40 during proteolysis.
Journal of Virology 08/2011; 85(19):10021-30. · 5.40 Impact Factor
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ABSTRACT: Human enterovirus 71 (EV71) is the major pathogen that causes hand, foot and mouth disease that particularly affects young children. Growing hand, foot and mouth disease outbreaks were observed worldwide in recent years and caused devastating losses both economically and politically. However, vaccines or effective drugs are unavailable to date. The genome of EV71 consists of a positive sense, single-stranded RNA of ∼7400 bp, encoding a large precursor polyprotein that requires proteolytic processing to generate mature viral proteins. The proteolytic processing mainly depends on EV71 3C protease (3C(pro)) that possesses both proteolysis and RNA binding activities, which enable the protease to perform multiple tasks in viral replication and pathogen-host interactions. The central roles played by EV71 3C(pro) make it an appealing target for antiviral drug development. We determined the first crystal structure of EV71 3C(pro) and analyzed its enzymatic activity. The crystal structure shows that EV71 3C(pro) has a typical chymotrypsin-like fold that is common in picornaviral 3C(pro). Strikingly, we found an important surface loop, also denoted as β-ribbon, which adopts a novel open conformation in EV71 3C(pro). We identified two important residues located at the base of the β-ribbon, Gly123 and His133, which form hinges that govern the intrinsic flexibility of the ribbon. Structure-guided mutagenesis studies revealed that the hinge residues are important to EV71 3C(pro) proteolytic activities. In summary, our work provides the first structural insight into EV71 3C(pro), including a mobile β-ribbon, which is relevant to the proteolytic mechanism. Our data also provides a framework for structure-guided inhibitor design against EV71 3C(pro).
Journal of Molecular Biology 03/2011; 408(3):449-61. · 4.00 Impact Factor
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ABSTRACT: Androgens are known to modulate the skeletal muscle proliferation and differentiation processes. Recent in vitro studies have shown that dihydrotestosterone and anabolic steroids have functions in promoting the proliferation and differentiation of the mouse skeletal muscle myoblast C2C12 cell line through the classical androgen receptor (AR) signaling pathway. But there are contradictory reports that androgen plays its roles through the membrane signaling pathways. In the present study, we show that there is no expression of the classical AR in L6 cells both at gene and protein levels. We then investigated the effects of testosterone (T) on L6 cell proliferation and differentiation. The results show that T promotes L6 cell proliferation after a 24 h treatment, which followed by enhancing L6 cell differentiation, but these effects are not inhibited by flutamide (F), an antagonist of intracellular AR. Further, we tested the effect of testosterone covalently bounding to albumin (T-BSA), which does not cross the plasma membrane. The results demonstrate that T-BSA and free T have similar effects on L6 cell proliferation and differentiation, and that these effects involve G protein-coupled receptors and different downstream pathways. The L6 cell proliferation induced by T involves PKC and ERK1/2 signaling pathways and cell differentiation happens via the PKA signaling pathway. These results suggest that T promotes cell proliferation and differentiation via G protein-coupled receptors and different downstream pathways in the L6 cell line, although the related molecular mechanisms need to be elucidated in future studies.
Journal of Cellular Physiology 03/2011; 227(1):98-107. · 3.87 Impact Factor
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ABSTRACT: The Lin-11, Isl-1, and Mec-3 (LIM) homeodomain transcription factor Isl-1 has been reported to be involved in pituitary development in the early stages of mouse embryogenesis. Our recent studies have shown that Isl-1 is mainly located in the pituitary gonadotropes throughout pituitary development and persists to adulthood. We still do not know the physiological functions of Isl-1 expression and its related mechanisms in the pituitary gland. The aim of the present study was to examine the hypothesis that Isl-1 is involved in regulating pituitary gonadotropin hormone (FSH/LH) production by activating FSHβ and LHβ gene expressions. We have shown that Isl-1 activates FSHβ and LHβ subunit promoters and endogenous gene transcription in LβT2 cells. In addition, Isl-1 overexpression significantly increased FSH synthesis and secretion but not LH. The actions of Isl-1 were not observed when the homeodomain or LIM1 domains are mutated. This demonstrates that Isl-1 induction of FSHβ and LHβ is by both direct and indirect binding of Isl-1 to DNA sequences. Furthermore, Isl-1 expressional level was up-regulated in LβT2 cells after exposure to GnRH, activin, and leptin. However, RNA interference-induced knockdown of Isl-1 significantly reduced the effect of leptin but did not obviously influence the stimulating effects of GnRH and activin on LH and FSH production. In conclusion, the results demonstrate that the LIM-homeodomain transcription factor Isl-1 functions to increase FSHβ/LHβ gene transcription, and mediates the effects of leptin on gonadotropin synthesis.
Endocrinology 10/2010; 151(10):4787-800. · 4.46 Impact Factor
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ABSTRACT: In the adult rat, the superior spermatic nerve (SSN) and inferior spermatic nerve (ISN) are involved in regulating testosterone secretion and spermatogenesis, in addition to endocrine control mechanisms. However, there are currently few data on how the testis nerve supply regulates testicular development and related mechanisms. The present study was thus designed to investigate the regulating effects of testis nerve supply to testicular maturation, spermatogenesis and the involved mechanisms from prepuberty to adulthood in rats. We transected the SSNs and ISNs of rats on postnatal day (PD) 30 and then analyzed changes in testicular morphology and cauda epididymal sperm content, cell proliferation and apoptosis and primary spermatocyte meiosis on PD60 and PD90. The results demonstrated that testicular denervation significantly reduced testis mass, cauda epididymal sperm counts and serum testosterone concentrations. Proliferating cell nuclear antigen (PCNA) and cleaved caspase-3 immunohistochemistry staining proved that the denervation had no influence on the proliferation of spermatogonia and primary spermatocytes, but obviously promoted the apoptosis of round spermatids and Leydig cells. It is novel that denervation reduced the meiotic activation of zygotene and pachytene spermatocytes through the expression of synaptonemal complex protein 3 (SCP3)-a marker of meiosis. In addition, RT-PCR showed that testis denervation significantly decreased testis 3beta-hydroxysteroid dehydrogenase 1 (3beta-HSD1) and luteinizing hormone receptor (LHR) mRNA levels, but had no obvious influence on testis follicle stimulating hormone receptor (FSHR) mRNA expression. These results suggest that the testicular nerve supply plays an important role in supporting seminiferous tubule development and spermatogenesis from prepuberty to adulthood.
Journal of Reproduction and Development 08/2010; 56(4):370-8. · 1.46 Impact Factor
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ABSTRACT: Although researchers have recently begun to pay more attention to the immunological characteristics of microvascular endothelial cells (MVECs), there are no reports on whether activation of MVECs by interferon-γ (IFN-γ) exerts any influence on the expressions of IFN-α/β. In the present study, we examined the influence of IFN-γ on the expressions of IFN-α/β in rat intestinal mucous MVECs (RIMMVECs). Different concentrations of IFN-γ were used to stimulate cultured RIMMVECs in vitro, and the cells and cell supernatants were collected at different time intervals. The influence of IFN-γ on the expressions of IFN-α/β in the RIMMVECs was examined at the mRNA and protein levels by real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The results indicated that IFN-γ was able to activate RIMMVECs, thereby leading to upregulated expressions of IFN-α/β. The real-time quantitative PCR analyses indicated that the IFN-α/β mRNA expression levels in RIMMVECs achieved their peak values after stimulation with IFN-γ at 20 ng/mL for 6 h and were increased by 14.88- and 3.82-fold, respectively, when compared with the levels in negative control cells. The ELISA analyses revealed that the IFN-α/β protein expression levels achieved their peak values after stimulation with IFN-γ at 40 ng/mL. The expression of IFN-α protein achieved its peak value at 12 h, while the expression of IFN-β protein achieved its peak value after 6 h. The present results suggest that the expression and secretion of IFNs may participate in the immunologic barrier function of MVECs.
Immunopharmacology and Immunotoxicology 03/2010; 32(4):656-62. · 1.83 Impact Factor
