Discoidin domain receptor 2 expression increases phagocytotic capacity in sertoli cells of sertoli cell-only syndrome testes.

Discoidin domain receptor 2 (DDR2) belongs to the receptor tyrosine kinase (RTK) family, other RTKs have been reported to regulate phagocytic function of Sertoli cells (SCs), yet little is known about the function of DDR2 in Sertoli cells. In the present study, we aim to explore the function and mechanism of ectopic discoidin domain receptor 2 (DDR2) expression in Sertoli cells of Sertoli cell-only syndrome (SCOS) testes. We found that discoidin domain receptor 2 (DDR2) was absent in Sertoli cells of normal testis but was expressed in Sertoli cells of SCOS testes. This Sertoli cell DDR2 expression was induced by impaired androgen receptor (AR) signaling, but was inhibited by increased AR signaling from testosterone administration. The Sertoli cell DDR2 expression led to an increase in phagocytosis through up-regulation of Scavenger receptor class B member 1 (SR-BI) levels. However, loss of DDR2 by knock-out or knock-down weakened the phagocytotic capacity of Sertoli cells. Furthermore, the expression of DDR2 in Sertoli cells activated matrix metallopeptidase 9 (MMP-9) to consume abnormal collagen increase in seminiferous tubules which was responsible for the block of testosterone transportation and AR loss and to compensate for the impaired blood-testis-barrier (BTB). Our data suggest that the AR/DDR2 cascade may serve as a negative feedback mechanism to help compensate for the homeostasis of seminiferous epithelium in SCOS testis.

Targeting of Discoidin Domain Receptor 2 (DDR2) Prevents Myofibroblast Activation and Neovessel Formation During Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal human disease with short survival time and few treatment options. Herein, we demonstrated that discoidin domain receptor 2 (DDR2), a receptor tyrosine kinase that predominantly transduces signals from fibrillar collagens, plays a critical role in the induction of fibrosis and angiogenesis in the lung. In vitro cell studies showed that DDR2 can synergize the actions of both transforming growth factor (TGF)-ß and fibrillar collagen to stimulate lung fibroblasts to undergo myofibroblastic changes and vascular endothelial growth factor (VEGF) expression. In addition, we confirmed that late treatment of the injured mice with specific siRNA against DDR2 or its kinase inhibitor exhibited therapeutic efficacy against lung fibrosis. Thus, this study not only elucidated novel mechanisms by which DDR2 controls the development of pulmonary fibrosis, but also provided candidate target for the intervention of this stubborn disease.

N-myc downstream-regulated gene 4, up-regulated by tumor necrosis factor-α and nuclear factor kappa B, aggravates cardiac ischemia/reperfusion injury by inhibiting reperfusion injury salvage kinase pathway.

N-myc downstream-regulated gene 4 (NDRG4) is expressed weakly in heart and has been reported to modulate cardiac development and QT interval duration, but the role of NDRG4 in myocardial ischemia/reperfusion (I/R) injury remains unknown. In the present study, we analyzed the expression as well as potential function of cardiac NDRG4 and investigated how NDRG4 expression is regulated by inflammation. We found that NDRG4 was weakly expressed in cardiomyocytes and that its expression increased significantly both in I/R injured heart and in hypoxia-reoxygenation (H/R) injured neonatal rat ventricular myocytes (NRVMs). The increased NDRG4 expression aggravated myocardial I/R injury by inhibiting the activation of the reperfusion injury salvage kinase (RISK) pathway. Forced over-expression of NDRG4 inhibited RISK activation and exacerbated injury not only in I/R injured heart, but also in H/R treated NRVMs, whereas short hairpin RNA (shRNA)-mediated knock-down of NDRG4 enhanced RISK activation and attenuated injury. Upon injury, myocardial NDRG4 expression was induced by tumor necrosis factor-α (TNF-α) through nuclear factor kappa B (NF-κB), and we found that pre-treatment with inhibitors of either TNF-α or NF-κB blocked NDRG4 expression as well as I/R injury in vivo and H/R injury in vitro. Our study indicates that up-regulation of NDRG4 aggravates myocardial I/R injury by inhibiting activation of the RISK pathway, thereby identifying NDRG4 as a potential therapeutic target in I/R injury.

Abnormal Accumulation of Collagen Type I Due to the Loss of Discoidin Domain Receptor 2 (Ddr2) Promotes Testicular Interstitial Dysfunction.

BACKGROUND: Loss of functional allele for discoidin domain receptor 2 (Ddr2) results in impaired Leydig cell response to luteinizing hormone (LH), low testosterone production and arrested spermatogenesis in older male Ddr2slie/slie mice. However, the underlying mechanism responsible for this phenotype remains unknown. Herein, we reported for the first time that the deregulated expression of Ddr2 cognate ligand, namely collagen type I (COL1), may account for the disruption of the testicular steroidogenesis in Ddr2slie/slie mutant testes. METHODOLOGY/PRINCIPAL FINDINGS: Expression of Ddr2 increased gradually along postnatal development, whereas COL1 expression became negligible from adulthood onwards. In Ddr2slie/slie mutant testis, however, in contrast to the undetectable staining of Ddr2, COL1 expression was constantly detected, with the highest values detected during adulthood. In the experimental vasectomy model, Ddr2slie/slie mutant mice exhibited an early androgen deficiency than wild-type mice, along with the accumulation of fibrotic tissue in the interstitium. Functionally, ablation of endogenous Ddr2 resulted in a significant decrease of testosterone (T) level in TM3 cells in the presence of higher concentration of COL1 treatment. Conversely, overexpression of Ddr2 could help TM3 cells to maintain a normal testicular steroidogenesis even in the presence of high concentration of COL1. Additionally, attenuated expression of Ddr2 correlates to the deregulated level of serum T levels in human pathological testes. CONCLUSIONS: Abnormal accumulation of interstitial COL1 may be responsible for the steroidogenic dysfunction in Ddr2slie/slie mutant testes.

Overexpression of DDR2 contributes to cell invasion and migration in head and neck squamous cell carcinoma.

Background Discoidin domain receptor 2 (DDR2) is a unique receptor tyrosine kinase (RTK) that is activated by fibrillar collagens. Although DDR2 contributes to the metastasis of some tumors, its role in head and neck squamous cell carcinoma (HNSCC) remains unknown. The aim of this study was to investigate the expression level, clinical and pathological significance, and biologic function of DDR2 in HNSCC. Methods Real-time quantitative PCR, western blot, and immunohistochemical staining were employed to assess the expression levels of DDR2 in HNSCC specimens. Adenovirus-mediated overexpression of DDR2 was used to evaluate its consequences on cell proliferation, invasion, migration, and the process of hypoxia-induced epithelial-mesenchymal transition (EMT). Then nude mouse xenograft and tail vein metastasis models were utilized to validate the in vitro results. Results DDR2 was highly expressed in high grade HNSCC tissues and lowly expressed in low grade HNSCC tissues, but absent or rarely expressed in cancer-associated normal tissues. Both the frequency and expression intensity of DDR2 were significantly associated with tumor pathologic stage and lymph node metastasis. In vitro, DDR2 overexpression in HNSCC cells failed to alter cell proliferation but markedly accelerates cell invasion and migration as well as hypoxia-induced EMT. In vivo, elevated expression of DDR2 speeds up the metastasis of HNSCC cells to the lung. Conclusion DDR2 plays an important role in HNSCC metastasis, and might be a promising target for future therapies in this type of cancer.

A host deficiency of discoidin domain receptor 2 (DDR2) inhibits both tumour angiogenesis and metastasis.

Discoidin domain receptor 2 (DDR2) is a unique receptor tyrosine kinase (RTK) that signals in response to collagen binding and is implicated in tumour malignant phenotypes such as invasion and metastasis. Although it has been reported that DDR2 expression is up-regulated in activated endothelial cells (ECs), functional studies are lacking. Herein, we found that enforced expression of DDR2 promoted proliferation, migration and tube formation of primary human umbilical vein endothelial cells (HUVECs). The results of immunohistochemical analysis showed a strikingly high level of DDR2 in human tumour ECs. Most significantly, we discovered that a host deficiency of DDR2 inhibits subcutaneous angiogenesis induced by either VEGF or tumour cells. In addition, the remaining tumour vessels in DDR2-deficient mice exhibit some normalized properties. These vascular phenotypes are accompanied by the up-regulation of anti-angiogenic genes and down-regulation of pro-angiogenic genes, as well as by alleviated tumour hypoxia. By use of a tail vein metastasis model of melanoma, we uncovered that loss of stromal DDR2 also suppresses tumour metastasis to the lung. Hence, our current data disclose a new mechanism by which DDR2 affects tumour progression, and may strengthen the feasibility of targeting DDR2 as an anticancer strategy.

Expression of Rap1 during germ cell development in the rat and its functional implications in 2-methoxyacetic acid-induced spermatocyte apoptosis.

OBJECTIVE: To investigate the expression pattern of Ras-related protein 1 (Rap1) during testicular development and to clarify whether its expression is developmentally regulated and whether this expression is involved in the process of germ cell apoptosis. MATERIALS AND METHODS: The expression pattern of Rap1 in the adult rat testicle was compared with that in the developing rat testicle using immunoblotting and immunohistochemical analyses. After the adult rats were treated with methoxyacetic acid (MAA), which selectively depletes primary spermatocytes, we correlated Rap1 expression with apoptotic dynamics using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL), double immunofluorescent staining, and coimmunoprecipitation assays. RESULTS: During testicular development, Rap1 was expressed in the nucleus of gonocytes and in the Golgi apparatus of spermatocytes. The expression pattern of Rap1 during spermatogenesis was also shown to be stage specific. After 12 hours of MAA treatment, we found that Rap1 was translocated into the nucleus of some spermatocytes and the overlapping rate of Rap1 and NF-κB was much greater than that of Rap1 and TUNEL staining. In addition, Rap1 protein could be co-immunoprecipitated with NF-κB protein. CONCLUSION: In cooperation with NF-κB, Rap1 might be involved in the early stage of the apoptotic process occurring in the MAA-treated rat testicle. Additional characterization of this small guanosine triphosphatase in the apoptotic dynamics should provide information on the early diagnosis of MAA-induced male infertility.

Presence of metastasis-associated protein 1 in Sertoli cells is required for proper contact between Sertoli cells and adjacent germ cells.

OBJECTIVE: To investigate whether the normal expression of metastasis-associated protein 1 (MTA1) in Sertoli cells (SCs) is associated with adjacent germ cells (GCs) and to provide the functional relevance of MTA1 in this somatic cell. METHODS: The expression pattern of MTA1 in the SCs of impaired human spermatogenesis was determined using immunohistochemistry. The effect of the depletion of GCs on the expression of MTA1 in isolated SCs was evaluated using reverse transcriptase polymerase chain reaction in murine testes treated with busulphan. Finally, using multiple assays, the functional investigation of MTA1 by its specific knockdown was performed in SC-GC co-cultures. RESULTS: SCs were negatively immunolabeled in the tubules with impaired spermatogenesis. Depletion of murine GCs by treatment with busulphan resulted in a dramatic decrease of the MTA1 transcripts level in the isolated SCs on the 15th day of treatment and thereafter had totally abolished MTA1 expression by the 30th day of treatment, respectively. The addition of isolated round spermatids into SC culture could partially elevate MTA1 expression in the latter. Furthermore, MTA1 is crucial to maintain the GC nursery function and normal anchoring junction formation in SCs because ablation of MTA1 by siRNA induced extensive defects of genes related to SC homeostasis. CONCLUSION: We propose a novel role for SC-expressing MTA1, which is determined by the presence of surrounding GCs, in mediating the crosstalk between SCs and GCs by influencing a broad spectrum of gene changes.

Inhibition of ghrelin signaling improves the reproductive phenotype of male ob/ob mouse.

OBJECTIVE: To investigate whether ghrelin signaling is involved in the pathogenesis of male factor infertility induced by leptin deficiency. DESIGN: Experimental study. SETTING: University academic medical center. ANIMAL(S): Ten-week-old C57BL/6J mice and ob/ob mice. INTERVENTION(S): Western blotting, (quantitative) reverse transcription-polymerase chain reaction (qRT-PCR), immunohistochemistry, and in situ end labeling of fragmented DNA. MAIN OUTCOME MEASURE(S): Expression levels of ghrelin and its functional receptor growth hormone (GH) secretagogue receptor 1a (GHS-R1α) were examined by Western blotting and immunohistochemistry. Ob/ob mice were injected IP with specific GHS-R1α antagonist, and thereafter germ cell apoptosis and steroidogenic capability were assessed by TUNEL assay, (q) RT-PCR, and radioimmunoassay. RESULT(S): Expression of GHS-R1α and its endogenous ligand ghrelin was both up-regulated in ob/ob testis. Inhibition of the ghrelin pathway restored androgen synthesis, reduced germ cell apoptosis, and thereby resulted in improved sperm production in ob/ob mice. CONCLUSION(S): Ghrelin, as an antagonistic partner of leptin in the endocrinic/paracrine circuit, may be involved in the pathogenesis of male factor infertility induced by leptin deficiency.

Sertoli cell-specific expression of metastasis-associated protein 2 (MTA2) is required for transcriptional regulation of the follicle-stimulating hormone receptor (FSHR) gene during spermatogenesis.

BACKGROUND: Desensitization of FSH response by down-regulation of FSHR transcription is critical for FSH action. RESULTS: Chromatin modifier MTA2 participates in the down-regulation of FSHR transcription. CONCLUSION: The FSH/Ar/MTA2 cascade may serve as an indispensable negative feedback mechanism to modulate FSH transduction events in Sertoli cells. SIGNIFICANCE: Our findings provide new insights into mechanisms by which FSH is deregulated in male infertile patients. The effect of follicle-stimulating hormone (FSH) on spermatogenesis is modulated at a fundamental level by controlling the number of competent receptors present at the surface of Sertoli cells (SCs). One underlying mechanism is the down-regulation of the expression levels of the FSH receptor (FSHR) gene after exposure to FSH. Here we report that metastasis-associated protein 2 (MTA2), a component of histone deacetylase and nucleosome-remodeling complexes, as a gene product induced directly by testosterone or indirectly by FSH, is exclusively expressed in SCs. Stimulation of SCs with FSH is accompanied by up-regulation of MTA2 expression and enhancement of deacetylase activity. This effect requires the integrity of functional androgen receptor. Furthermore, MTA2 is a potent corepressor of FSHR transcription, because it can recruit histone deacetylase-1 onto the FSHR promoter and participates in the down-regulation of FSHR expression upon FSH treatment. Abolishment of endogenous MTA2 by siRNA treatment disrupted the desensitization of the FSH response and thereafter impaired the FSH-dependent secretory function of SCs. From a clinical standpoint, deregulated expression of MTA2 in SCs of human pathological testes negatively correlates to the deregulated level of serum FSH. Overall, our present results provide the first evidence that the FSH/androgen receptor/MTA2 cascade may serve as an indispensable negative feedback mechanism to modulate the transduction events of SCs in response to FSH. These data also underscore an unexpected reproductive facet of MTA2, which may operate as a novel integrator linking synergistic actions of FSH and androgen signaling in SCs.

Up-regulation of NDRG2 through nuclear factor-kappa B is required for Leydig cell apoptosis in both human and murine infertile testes.

Many pro-apoptotic factors, such as nuclear factor-kappa B (NF-κB) and Fas, play crucial roles in the process of Leydig cell apoptosis, ultimately leading to male sterility, such as in Sertoli cell only syndrome (SCO) and hypospermatogenesis. However, the molecular mechanism of such apoptosis is unclear. Recent reports on N-myc downstream-regulated gene 2 (ndrg2) have suggested that it is involved in cellular differentiation, development, and apoptosis. The unique expression of NDRG2 in SCO and hypospermatogenic testis suggests its pivotal role in those diseases. In this study, we analyzed NDRG2 expression profiles in the testes of normal spermatogenesis patients, hypospermatogenesis patients, and SCO patients, as well as in vivo and in vitro models, which were Sprague-Dawley rats and the Leydig cell line TM3 treated with the Leydig cell-specific toxicant ethane-dimethanesulfonate (EDS). Our data confirm that NDRG2 is normally exclusively located in the cytoplasm of Leydig cells and is up-regulated and translocates into the nucleus under apoptotic stimulations in human and murine testis. Meanwhile, transcription factor NF-κB was activated by EDS administration, bound to the ndrg2 promoter, and further increased in expression, effects that were abolished by NF-κB inhibitor Pyrrolidine dithiocarbamate (PDTC). Furthermore, siRNA knock-down of ndrg2 led to increased proliferative or decreased apoptotic TM3 cells, while over-expression of ndrg2 had the reverse effect. This study reveals that ndrg2 is a novel gene that participates in Leydig cell apoptosis, with essential functions in testicular cells, and suggests its possible role in apoptotic Leydig cells and male fertility.