Yuanqiang Zhang

Expression analysis of the NDRG2 gene in mouse embryonic and adult tissues

N-myc downstream-regulated gene 2 (NDRG2) is believed to be involved in cell growth events. However, its exact function is still unknown. To elucidate the role of this gene, we used an anti-Ndrg2 monoclonal antibody in immunohistochemistry and immunofluorescence assays to analyze the expression pattern of Ndrg2 protein in mouse embryos at various gestational ages and in a variety of adult mouse tissues. Ndrg2 immunoreactivity was generally localized to the cytoplasm. During mouse development, Ndrg2 expression was observed in many developing tissues and organs including the heart, brain, lung, gut, liver, kidney, skeletal muscle, cartilage, chorion, epidermis, and whisker follicles. Ndrg2 expression was developmentally dynamic, being generally lower in the early stages of development and markedly increasing during later stages. Ndrg2 expression was also observed in a variety of adult mouse tissues, particularly in the heart and brain. This is the first demonstration of Ndrg2 protein expression in both embryonic and adult mouse tissues. Our results suggest that NDRG2 plays important roles in histogenesis and organogenesis.

Transient protection from heat-stress induced apoptotic stimulation by metastasis-associated protein 1 in pachytene spermatocytes.

Background Deregulated thermal factors have been frequently implicated in the pathogenesis of male infertility, but the molecular basis through which certain responses are directed remain largely unknown. We previously reported that overexpression of exogenous Metastasis-associated protein 1 (MTA1) protects spermatogenic tumor cells GC-2spd (ts) against heat-induced apoptosis. To further dissect the underlying mechanism, we addressed here the fine coordination between MTA1 and p53 in pachytene spermatocytes upon hyperthermal stimulation. Methodology/Principal Findings High level of MTA1 expression sustained for 1.5 h in primary spermatocytes after heat stress before a notable decrease was detected conversely correlated to the gradual increase of acetylation status of p53 and of p21 level. Knockdown of the endogenous MTA1 in GC-2spd (ts) elevated the acetylation of p53 by diminishing the recruitment of HDAC2 and thereafter led to a dramatic increase of apoptosis after heat treatment. Consistent with this, in vivo interference of MTA1 expression in the testes of C57BL/6 mice also urged an impairment of the differentiation of spermatocytes and a disruption of Sertoli cell function due to the elevated apoptotic rate after heat stress. Finally, attenuated expression of MTA1 of pachytene spermatocytes was observed in arrested testes (at the round spermatid level) of human varicocele patients. Conclusions These data underscore a transient protective effect of this histone modifier in primary spermatocytes against heat-stress, which may operate as a negative coregulator of p53 in maintenance of apoptotic balance during early phase after hyperthermal stress.

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.

Discoidin domain receptor 2 is associated with the increased expression of matrix metalloproteinase-13 in synovial fibroblasts of rheumatoid arthritis

Regulation of matrix metalloproteinase-13 (MMP-13) by collagen matrix in the synovial fibroblasts of rheumatoid arthritis (RA) is critical event in the progressive joint destruction. Our previous study indicated that a collagen receptor, discoidin receptor 2 (DDR2), was highly expressed in the synovial fibroblasts of RA. However, the functional role of DDR2 in the regulation of MMP-13 production in synovial fibroblasts has not been elucidated. In this study, we initially demonstrated that the DDR2 and MMP-13 proteins are both highly expressed in the synovial lining layer of RA. MMP-13 mRNA and protein in synovial fibroblasts of RA were preferentially induced by collagen type II compared with MMP-1. Furthermore, stable overexpression of wild type DDR2 in murine synoviocytes dramatically augments the production of MMP-13. The activation of DDR2 also mediates the up-regulation of MMP-13 promoter activity in 293T cells. Inhibitor specific for extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) cascade was shown to decrease MMP-13 level induced by collagen II in RA synovial fibroblasts and DDR2-induced MMP-13 promoter activity. Runx2 and activator protein-1 (AP-1) binding sites in MMP-13 promoter region are required for DDR2-induced transcription. The data in this study suggest that DDR2-mediated MMP-13 induction by collagen matrix in synovial fibroblasts of RA contributed to articular cartilage destruction.

Metastasis tumor antigen 1 is involved in the resistance to heat stress‐induced testicular apoptosis

Our previous study documented the expression of Mta1 during spermatogenesis. Here, we present evidence for a possible involvement of Mta1 in the regulation of testicular function, possibly by interacting with p53. A notable decrease of Mta1 expression was revealed at postsurgical day 6, consistent with the previously reported upregualtion of p53 in mouse cryptorchidism. Furthermore, in vitro over‐expression of Mta1 could remarkably elevate the resistance capability of spermatogenic tumor cells against heat‐induced apoptosis with a marked impairment of p53 expression. These findings indicate that Mta1 may operate as a negative modifier of apoptosis by interacting with p53 during gametogenesis.

Expression profile of MTA1 in adult mouse tissues

MTA1, as a constituent of the nucleosome-remodeling and -deacetylation complex (NuRD), is thought to modulate transcription by influencing the status of chromatin remodeling. Despite its strong correlation with the metastatic potential of several cancer cell lines and tissues, MTA1 can also regulate divergent cellular pathways by modifying the acetylation status of crucial target genes. However, its fundamental physiological functions have not been characterized. To further address the possible physiological role of this protein in mammals, the authors examined the expression pattern of mouse MTA1 in a variety of adult mouse tissues by a combination of techniques, including semi-quantitative RT-PCR, Western blotting and immunohistochemistry. Positive signals were observed on variety of tissues/cells in multiple systems including nervous, cardiovascular, respiratory, digestive, immune, endocrine, urinary, reproductive and sensory organ systems. MTA1 was localized in both the cytoplasm and the nuclei, and was accumulated in the nuclei. In mature mice, MTA1 expression was seen in cell types that constantly undergo proliferation or self-renewal, such as testis and cell types not constantly engaged in proliferation or self-renewal, such as brain, liver and kidney. This differential expression suggests that this protein serves distinct functions in murine organs.

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.

NDRG2 is highly expressed in pancreatic β cells and involved in protection against lipotoxicity

The N-myc downstream-regulated gene 2 (NDRG2) is involved in cell differentiation and apoptosis, but its function in the pancreas remains to be established. Herein we examine the expression and function of NDRG2 in the endocrine pancreas. NDRG2 immunoreactivity was localized mainly in the cytoplasm of pancreatic β cells. When β-TC3 cells were exposed chronically to high levels of free fatty acid (FFA), cell viability was impaired, and Akt and NDRG2 phosphorylation were reduced. NDRG2 is a potential substrate of protein kinase Akt. Overexpression of constitutively active Akt enhanced NDRG2 phosphorylation and abolished the apoptosis induced by FFA in β-TC3 cells, whereas NDRG2 knock-down attenuated Akt-mediated protection of β cells against fatty acid-triggered apoptosis. Collectively, these data indicate that NDRG2 acts as a key molecule in pancreatic β cells and is involved in the Akt-mediated protection of β cells against lipotoxicity.

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.

Involvement of metastasis tumor antigen 1 in hepatic regeneration and proliferation.

Background/Aims: Metastasis tumor antigen 1 (MTA1), an integral part of nucleosome remodeling and histone deacetylation (NuRD) complexes, is well correlated with the potential of metastasis, with the ability to regulate divergent cellular pathways by modifying the acetylation status of crucial target genes. However, additional biological functions of this molecule remain largely unexplored. This study was undertaken to explore the potential role of this molecule in mouse liver. Methods: MTA1 expression was firstly explored in mouse partial hepatectomy model (PHx). The effect of overexpression of MTA1 on hepatic proliferation and differentiation was then examined in vivo by hydrodynamic-based gene transfer method and in vitro using transformed cell line AML12 overexpressing MTA1, respectively. Results: Consistent with the hepatic regeneration, MTA1 expression was significantly increased 24h post-PHx, with a maximum level at 48h after PHx. MTA1 immunoreactivity was generally elevated right after PHx and the staining appeared to experience a cytoplasm-to-nuclear transition. Overexpression of exogenous MTA1 could notably stimulate hepatic proliferation in vivo and could also accelerate hepatocyte differentiation in vitro. Conclusion: These data underscore a hepatocelluar facet of this recently defined molecule, which may represent as a novel regulator and a new therapeutic target for the treatment of impaired liver.