Advancements in the ERK1/2 Signaling Pathway Affecting Male Reproduction.

Male infertility, age-related changes, and tumors have been increasingly studied in the field of male reproductive health due to the emergence of environmental stressors, declining fertility rates, and aging populations. Numerous studies have demonstrated that the ERK1/2 signaling pathway plays a significant role in male reproduction. The ERK1/2 pathway is associated with several signaling pathways and has a complex interplay that influences the spermatogenic microenvironment, sperm viability, gonadal axis regulation, as well as resistance to testicular aging and tumors. Moreover, the ERK1/2 pathway directly or indirectly regulates testicular somatic cells, which are crucial for maintaining spermatogenesis and microenvironment regulation. Given the critical role of the ERK1/2 signaling pathway in male reproductive health, comprehensive exploration of its multifaceted effects on male reproduction and underlying mechanisms is necessary. This study aims to provide a solid foundation for in-depth research in the field of male reproduction and further enhance the reproductive health of males.

Revealing the Mechanisms of Byu dMar 25 in the Treatment of Alzheimer's Disease through Network Pharmacology, Molecular Docking, and In Vivo Experiment.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease, severely reducing the cognitive level and life quality of patients. Byu dMar 25 (BM25) has been proved to have a therapeutic effect on AD. However, the pharmacological mechanism is still unclear. Therefore, this study aims to reveal the potential mechanism of BM25 affecting AD from the perspective of network pharmacology and experimental validation. METHODS: The potential active ingredients of BM25 were obtained from the TCMSP database and literature. Possible targets were predicted using SwissTargetPrediction tools. AD-related genes were identified by using GeneCards, OMIM, DisGeNET, and Drugbank databases. The candidate genes were obtained by extraction of the intersection network. Additionally, the "drug-target-disease" network was constructed by Cytoscape 3.7.2 for visualization. The PPI network was constructed by the STRING database, and the core network modules were filtered by Cytoscape 3.7.2. Enrichment analysis of GO and KEGG was carried out in the Metascape platform. Ledock software was used to dock the critical components with the core target. Furthermore, protein levels were evaluated by immunohistochemistry. RESULTS: In this study, 112 active components, 1112 disease candidate genes, 3084 GO functions, and 277 KEGG pathways were obtained. Molecular docking showed that the effective components of BM25 in treating AD were ß-asarone and hydroxysafflor yellow A. The most important targets were APP, PIK3R1, and PIK3CA. Enrichment analysis indicated that the Golgi genetic regulation, peroxidase activity regulation, phosphatidylinositol 3-kinase complex IA, 5-hydroxytryptamine receptor complexes, cancer pathways, and neuroactive ligand-receptor interactions played vital roles against AD. The rat experiment verified that BM25 affected PI3K-Akt pathway activation in AD. CONCLUSIONS: This study reveals the mechanism of BM25 in treating AD with network pharmacology, which provides a foundation for further study on the molecular mechanism of AD treatment.

Use of mouse hematopoietic stem and progenitor cells to treat acute kidney injury.

New and effective treatment for acute kidney injury remains a challenge. Here, we induced mouse hematopoietic stem and progenitor cells (HSPC) to differentiate into cells that partially resemble a renal cell phenotype and tested their therapeutic potential. We sequentially treated HSPC with a combination of protein factors for 1 wk to generate a large number of cells that expressed renal developmentally regulated genes and protein. Cell fate conversion was associated with increased histone acetylation on promoters of renal-related genes. Further treatment of the cells with a histone deacetylase inhibitor improved the efficiency of cell conversion by sixfold. Treated cells formed tubular structures in three-dimensional cultures and were integrated into tubules of embryonic kidney organ cultures. When injected under the renal capsule, they integrated into renal tubules of postischemic kidneys and expressed the epithelial marker E-cadherin. No teratoma formation was detected 2 and 6 mo after cell injection, supporting the safety of using these cells. Furthermore, intravenous injection of the cells into mice with renal ischemic injury improved kidney function and morphology by increasing endogenous renal repair and decreasing tubular cell death. The cells produced biologically effective concentrations of renotrophic factors including VEGF, IGF-1, and HGF to stimulate epithelial proliferation and tubular repair. Our study indicates that hematopoietic stem and progenitor cells can be converted to a large number of renal-like cells within a short period for potential treatment of acute kidney injury.

Polycystin-2 and phosphodiesterase 4C are components of a ciliary A-kinase anchoring protein complex that is disrupted in cystic kidney diseases.

Polycystic kidney disease (PKD) is a genetic disorder that is characterized by cyst formation in kidney tubules. PKD arises from abnormalities of the primary cilium, a sensory organelle located on the cell surface. Here, we show that the primary cilium of renal epithelial cells contains a protein complex comprising adenylyl cyclase 5/6 (AC5/6), A-kinase anchoring protein 150 (AKAP150), and protein kinase A. Loss of primary cilia caused by deletion of Kif3a results in activation of AC5 and increased cAMP levels. Polycystin-2 (PC2), a ciliary calcium channel that is mutated in human PKD, interacts with AC5/6 through its C terminus. Deletion of PC2 increases cAMP levels, which can be corrected by reexpression of wild-type PC2 but not by a mutant lacking calcium channel activity. Phosphodiesterase 4C (PDE4C), which catabolizes cAMP, is also located in renal primary cilia and interacts with the AKAP150 complex. Expression of PDE4C is regulated by the transcription factor hepatocyte nuclear factor-1ß (HNF-1ß), mutations of which produce kidney cysts. PDE4C is down-regulated and cAMP levels are increased in HNF-1ß mutant kidney cells and mice. Collectively, these findings identify PC2 and PDE4C as unique components of an AKAP complex in primary cilia and reveal a common mechanism for dysregulation of cAMP signaling in cystic kidney diseases arising from different gene mutations.

HNF-1beta regulates transcription of the PKD modifier gene Kif12.

Hepatocyte nuclear factor-1beta (HNF-1beta) is a transcription factor that regulates gene expression in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1beta lead to a syndrome of inherited renal cysts and diabetes and are also a common cause of sporadic renal dysplasia. The full complement of target genes responsible for the functions of HNF-1beta, however, is incompletely defined. Using a functional genomics approach involving chromatin immunoprecipitation and promoter arrays, combined with gene expression profiling, we found that an HNF-1beta target gene in the kidney is kinesin family member 12 (Kif12), a gene previously identified as a candidate modifier gene in the cpk mouse model of polycystic kidney disease. Mutations of HNF-1beta inhibited Kif12 transcription in both cultured cells and knockout mice by altering co-factor recruitment and histone modification. Because kinesin-12 family members participate in orienting cell division, downregulation of Kif12 may underlie the abnormal planar cell polarity observed in cystic kidney diseases.

Mutations of HNF-1beta inhibit epithelial morphogenesis through dysregulation of SOCS-3.

Hepatocyte nuclear factor-1beta (HNF-1beta) is a Pit-1, Oct-1/2, Unc-86 (POU) homeodomain-containing transcription factor expressed in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1beta cause maturity-onset diabetes of the young, type 5 (MODY5), which is characterized by early-onset diabetes mellitus and congenital malformations of the kidney, pancreas, and genital tract. Knockout of HNF-1beta in the mouse kidney results in cyst formation. However, the signaling pathways and transcriptional programs controlled by HNF-1beta are poorly understood. Using genome-wide chromatin immunoprecipitation and DNA microarray (ChIP-chip) and microarray analysis of mRNA expression, we identified SOCS3 (suppressor of cytokine signaling-3) as a previously unrecognized target gene of HNF-1beta in the kidney. HNF-1beta binds to the SOCS3 promoter and represses SOCS3 transcription. The expression of SOCS3 is increased in HNF-1beta knockout mice and in renal epithelial cells expressing dominant-negative mutant HNF-1beta. Increased levels of SOCS-3 inhibit HGF-induced tubulogenesis by decreasing phosphorylation of Erk and STAT-3. Conversely, knockdown of SOCS-3 in renal epithelial cells expressing dominant-negative mutant HNF-1beta rescues the defect in HGF-induced tubulogenesis by restoring phosphorylation of Erk and STAT-3. Thus, HNF-1beta regulates tubulogenesis by controlling the levels of SOCS-3 expression. Manipulating the levels of SOCS-3 may be a useful therapeutic approach for human diseases induced by HNF-1beta mutations.

The interferon-stimulated gene factor 3 complex mediates the inhibitory effect of interferon-beta on matrix metalloproteinase-9 expression.

Matrix metalloproteinase-9 (MMP-9) displays a preference for a broad range of substrates including extracellular matrix proteins and cytokines. MMP-9 plays an important role in physiological processes, as well as in inflammatory diseases and numerous cancers. Interferon-beta is a pleiotropic cytokine with antiviral, antiproliferative and immunomodulatory activities. Interferon-beta positively regulates gene expression, predominantly through the Janus kinase-signal transducer and activator of transcription (STAT) pathway. However, little is known about the mechanisms used by interferon-beta to negatively regulate gene expression. In the present study, we show that interferon-beta inhibits MMP-9 gene expression at the transcriptional level. Using cell lines deficient in three components of the interferon-beta-activated interferon-stimulated gene factor 3 (ISGF3) complex (i.e. STAT-1, STAT-2 and interferon regulatory factor 9), the results of our study indicate that all three members are required for interferon-beta inhibition. Chromatin immunoprecipitation assays demonstrate that interferon-beta reduces recruitment of transcriptional activators and coactivators, such as nuclear factor kappa B p65, Sp1, CREB-binding protein and p300, to the MMP-9 promoter, and decreases the degree of histone acetylation at the MMP-9 promoter. This occurs in the absence of an association of the ISGF3 complex with the MMP-9 promoter. Taken together, these data define the role of interferon-beta and the ISGF3 members in suppressing MMP-9 gene expression.

Interferon-gamma-activated STAT-1alpha suppresses MMP-9 gene transcription by sequestration of the coactivators CBP/p300.

Interferon-gamma (IFN-gamma) is a pleiotropic cytokine involved in aspects of immune regulation, cell proliferation, and host defense mechanisms directed toward various cancers. Some of the biological functions of IFN-gamma are achieved through inhibition of gene expression, although the mechanisms by which IFN-gamma suppresses gene transcription are poorly understood. Herein, we demonstrate the molecular basis by which IFN-gamma mediates suppression of the matrix metalloproteinase-9 (MMP-9) gene. IFN-gamma-activated signal transducer and activator of transcription-1alpha (STAT-1alpha) suppresses MMP-9 gene transcription, which is dependent on phosphorylation of tyrosine 701 but not phosphorylation of serine 727. The coactivator cyclic AMP response element-binding protein-binding protein (CBP) is an important component of induction of MMP-9 gene transcription. IFN-gamma induces the in vivo association of STAT-1alpha and CBP and decreases the association of CBP to the MMP-9 promoter. IFN-gamma does not influence the stability of CBP nor does IFN-gamma affect chromatin-remodeling events on the MMP-9 promoter. IFN-gamma inhibits the assembly of the MMP-9 transcription complex by suppressing H3/H4 acetylation and inhibiting recruitment of Pol II to the MMP-9 promoter. These findings indicate that IFN-gamma/STAT-1alpha exert their inhibitory effects by affecting multiple aspects of MMP-9 gene transcription.

Induction of a Th2 immune response by co-administration of recombinant adenovirus vectors encoding amyloid beta-protein and GM-CSF.

Lines of experimental evidence indicate that induction of humoral immune responses in transgenic mouse models of Alzheimer disease (AD) by repeated injection of synthetic amyloid beta-protein (Abeta) is effective in prevention and clearance of deposits of Abeta aggregates in the brain of the mice. We have tested a non-injection modality whereby replication-defective adenovirus vectors encoding Abeta or the 99-amino acid carboxyl terminal fragment of Abeta precursor were intranasally administered to mice to elicit immune responses against Abeta. When mice were immunized only with the adenovirus vectors, immune responses against Abeta were negligible. By co-immunization with an adenovirus vector encoding granulocyte-macrophage colony stimulating factor (GM-CSF), the adenovirus vector encoding Abeta effectively elicited an immune response against Abeta. Immunoglobulin isotyping demonstrated a predominant IgG1 and IgG2b response, suggesting a Th2 anti-inflammatory type. Thus, adjuvantation is essential for induction of an immune response against Abeta by adenovirus-mediated nasal vaccination.

Brg-1 is required for maximal transcription of the human matrix metalloproteinase-2 gene.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases whose aberrant expression are correlated with tumor invasion and angiogenesis. The transcription factors Sp1, Sp3, and AP-2 are required for constitutive expression of MMP-2 in tumor cells; however, the regulatory mechanisms of MMP-2 expression are not well understood. We investigated the involvement of Brg-1, the ATPase subunit of the SWI/SNF complex, in human MMP-2 gene transcription. Reconstitution of Brg-1 enhances MMP-2 transcription in Brg-1-deficient SW-13 cells. Chromatin immunoprecipitation assay demonstrates that Brg-1 is required for recruitment of Sp1, AP-2, and polymerase II to the MMP-2 promoter, whereas the binding of Sp3 to the MMP-2 promoter is decreased upon Brg-1 reconstitution. Furthermore, Sp1 interacts with Brg-1 in vivo. Restriction enzyme accessibility assays indicate that accessibility of the MMP-2 promoter region is not changed in the absence or presence of Brg-1. These results illustrate the connection between the SWI/SNF complex and optimal expression of MMP-2 and highlight the critical function of Brg-1 in regulating the recruitment of Sp1, Sp3, AP-2, and polymerase II to the MMP-2 promoter.

Class II major histocompatibility complex transactivator (CIITA) inhibits matrix metalloproteinase-9 gene expression.

Matrix metalloproteinases (MMPs) are a family of structurally related proteins with the collective capability to degrade all components of the extracellular matrix. Although MMP-mediated degradation of the extracellular matrix occurs physiologically, numerous pathological conditions exhibit increased MMP levels and excessive matrix degradation. Previous work from our laboratory has shown that interferon-gamma inhibits MMP-9 expression in a manner dependent upon STAT-1alpha. Here we extend our previous observations and show that the class II major histocompatibility complex transactivator (CIITA), a transcriptional target of STAT-1alpha, is also capable of inhibiting MMP-9 expression. By using stable cell lines that inducibly express CIITA or various mutant forms of CIITA, we show that CIITA requires the ability to bind the CREB-binding protein (CBP) to effectively inhibit MMP-9 expression. Furthermore, we show that CIITA-mediated inhibition of the MMP-9 gene does not rely on the transcriptional capability of CIITA. These findings support a model wherein CIITA inhibits MMP-9 expression by binding to and sequestering CBP, which reduces the levels of CBP at the MMP-9 promoter, inhibits levels of acetylated histone 3 at the MMP-9 promoter, and subsequently inhibits MMP-9 expression.

17beta-Estradiol inhibits class II major histocompatibility complex (MHC) expression: influence on histone modifications and cbp recruitment to the class II MHC promoter.

Major histocompatibility complex (MHC) class II proteins are important for the initiation of immune responses and are essential for specific recognition of foreign antigens by the immune system. Regulation of class II MHC expression primarily occurs at the transcriptional level. The class II transactivator protein is the master regulator that is essential for both constitutive and interferon-gamma-inducible class II MHC expression. Estrogen [17beta-estradiol (17beta-E2)] has been shown to have immunomodulatory effects. In this study, we show that 17beta-E2 down-regulates interferon-gamma inducible class II MHC protein levels on brain endothelial cells, as well as other cell types (astrocytes, fibrosacroma cells, macrophages). The inhibitory effects of 17beta-E2 on class II MHC expression are not due to changes in class II transactivator mRNA or protein levels, rather, 17beta-E2 mediates inhibition at the level of class II MHC gene expression. We demonstrate that 17beta-E2 attenuates H3 and H4 histone acetylation and cAMP response element binding protein-binding protein association with the class II MHC promoter, suggesting that 17beta-E2 inhibits class II MHC expression by a novel mechanism involving modification of the histone acetylation status of the class II MHC promoter.