Cyclin D1 Promoter Research Articles

Use of modified human hemangioma tissue cultures and human umbilical vein endothelial cell cultures to gain mechanistic insights into imiquimod treatment for infantile hemangioma.

Infantile hemangiomas (IH) are a common entity encountered by dermatologists, otolaryngologists, and other surgeons. Oral propranolol is a mainstay of treatment for IH and is well-tolerated, though propranolol-refractory IH and other drug-related adverse events are documented and can limit its usage. There are few in vitro testing systems for putative treatment agents. To address this, we modified a tissue culture system for human hemangioma treatment testing to evaluate the treatment impact of the immune modifier, imiquimod. Human umbilical vein endothelial cells (HUVEC) and hemangioma cultures were treated with several concentrations of imiquimod followed by MTT assays, reporter gene assays, PCR, ELISA, and Western blotting for IL-8, VEGF, Cyclin D1, and IFNα and immunohistochemistry for Cyclin D1 and Ki-67. HUVEC showed acute decreases in IL-8, VEGF, and Cyclin D1 promoter activity and increases in IFNα mRNA after imiquimod treatment. Hemangioma samples showed no change in Ki-67 or Cyclin D1 staining after treatment with imiquimod after 27 d, with significantly increased IL-8 and VEGF. From this preliminary analysis, we discerned that hemangioma tissues can be grown in tissue culture and used for drug treatment studies. We also conclude acute and chronic modulation of cell cycle, angiogenesis factors, and immunostimulatory conditions may be associated with imiquimod mechanisms of action in hemangioma involution.

Bone Marrow Mesenchymal Stem Cells-Derived Extracellular Vesicle miR-208a-3p Alleviating Spinal Cord Injury via Regulating the Biological Function of Spinal Cord Neurons.

We aim to explore the potential mechanism of bone marrow mesenchymal stem cells-derived extracellular vesicles (BMSCs-Exo) in improving spinal cord injury (SCI). Thirty male 12-week specific pathogen-free (SPF) Sprague-Dawley (SD) rats were used to construct SCI model invivo. Ten male 12-week SPF SD rats were used to extract BMSCs. The Basso, Beattie, Bresnahan (BBB) score was used to evaluate the motor function of rats. Real-time fluorescence quantitative PCR (RT-PCR), western blot (WB), and double luciferase assay were used to explore the regulation between rno-miR-208a-3p and Cdkn1a (p21) in BMSCs. Primary spinal cord neurons were treated with lipopolysaccharide (100 ng/mL) for 30 min to mimic SCI in vitro. Compared with the model group (14 scores), BMSCs-Exo increased BBB score (19 scores) in SCI rats. Compared with the sham group, Cdkn1a was upregulated, whereas rno-miR-208a-3p was downregulated in the model group. However, compared with the model group, Cdkn1a was downregulated, whereas rno-miR-208a-3p was upregulated in the BMSCs-Exo group. In addition, rno-miR-208a-3p inhibited the expression of Cdkn1a via direct binding way. BMSCs-Exo-rno-miR-208a-3p promoted the proliferation of primary spinal neurons via inhibiting apoptosis in vitro. Moreover, BMSCs-Exo-rno-miR-208a-3p promoted cyclin D1, CDK6, and Bcl-2 and inhibited Bax expression in a cell model of SCI. In conclusion, BMSCs-Exo-carried rno-miR-208a-3p significantly protects rats from SCI via regulating the Cdkn1a pathway.

CARM1 drives triple-negative breast cancer progression by coordinating with HIF1A.

Coactivator-associated arginine methyltransferase 1 (CARM1) promotes the development and metastasis of estrogen receptor alpha (ERα)-positive breast cancer. The function of CARM1 in triple-negative breast cancer (TNBC) is still unclear and requires further exploration. Here, we report that CARM1 promotes proliferation, epithelial-mesenchymal transition, and stemness in TNBC. CARM1 is upregulated in multiple cancers and its expression correlates with breast cancer progression. Genome-wide analysis of CARM1 showed that CARM1 is recruited by hypoxia-inducible factor-1 subunit alpha (HIF1A) and occupy the promoters of CDK4, Cyclin D1, β-Catenin, HIF1A, MALAT1, and SIX1 critically involved in cell cycle, HIF-1 signaling pathway, Wnt signaling pathway, VEGF signaling pathway, thereby modulating the proliferation and invasion of TNBC cells. We demonstrated that CARM1 is physically associated with and directly interacts with HIF1A. Moreover, we found that ellagic acid, an inhibitor of CARM1, can suppress the proliferation and invasion of TNBC by directly inhibiting CDK4 expression. Our research has determined the molecular basis of CARM1 carcinogenesis in TNBC and its effective natural inhibitor, which may provide new ideas and drugs for cancer therapy.

Androgens Modulate Bcl-2 Agonist of Cell Death (BAD) Expression and Function in Breast Cancer Cells.

Androgen receptor (AR) expression in estrogen receptor-positive (ER+) breast cancer (BC) correlates with lower tumor grade and a better clinical outcome. Additionally, in normal mammary epithelium or ER+ BC preclinical models, androgens counteract basal/ER-dependent proliferation. Here, we report an additional mechanism, underlining the protective role exerted by AR. Specifically, the activation of intracellular AR upregulates the Bcl-2-family protein BAD, and TCGA database analyses show that in ER+ BC, BAD expression is associated with better disease-free survival. Ligand-activated AR influences its own and BAD cellular compartmentalization by enhancing levels in the nucleus, as well as in mitochondrial fractions. In both compartments, BAD exerts unconventional functions. In the nucleus, BAD and AR physically interact and, upon androgen stimulation, are recruited at the AP-1 and ARE sites within the cyclin D1 promoter region, contributing to explaining the anti-proliferative effect of androgens in BC cells. Androgens cause an enrichment in BAD and AR content in the mitochondria, correlated with a decrease in mitochondrial function. Thus, we have defined a novel mechanism by which androgens modulate BAD expression, its mitochondria localization, and nuclear content to force its ability to act as a cell cycle inhibitor, strengthening the protective role of androgen signaling in estrogen-responsive BCs.

The CRM1‐mediated nuclear export effectuates the role conversion of tumour suppressor gene BATF2 in colorectal cancer

The CRM1‐mediated nuclear export effectuates the role conversion of tumour suppressor gene BATF2 in colorectal cancer

Adipose Tissue-Mesenchymal Stem Cells Caused to Change the Methylation Status of hTERT Gene Promoter CpG Islands of Molt-4 Leukemia Cells as Cell-based Therapy.

DNA methylation was considered as prognostic information in some hematological malignancies. Previous studies have reported the in vitro and in vivo biology role of mesenchymal stem cells (MSCs) on leukemic cells. The aim of this study was to investigate the effect of MSCs on the promoter methylation status of hTERT as a catalytic subunit of telomerase enzyme. In the experimental study, the Molt-4 leukemic cells were co-cultured with MSCs for 7 days. At the end of the co-culture period, the Molt-4 cells were collected, DNA and protein were extracted. Then methylation specific-PCR and western blotting were done for evaluating the hTERT gene promoter methylation status and cyclin D1 and hTERT protein expression, respectively. In the following, the flow cytometry was done for cell cycle distribution assay. It was found that MSCs resulted in a significant decrease in the cyclin D1 and hTERT protein expression levels. Also, MSCs caused changes in the methylation status of the CpG islands in the hTERT gene promoter region. The following results showed that MSCs caused a significant increase in the number of cells at G0/G1 phase and arrest the G0/G1 phase as well as decrease in the cell proliferation of Molt-4 cells. It is concluded that co-culture of MSCs with Molt-4 cells could be involved in changing the methylation status of hTERT gene promoter, cell cycle and hTERT protein expression; it could be potentially beneficial for further investigations regarding the cell transplantation and cell-based therapy.

The SREBP-dependent regulation of cyclin D1 coordinates cell proliferation and lipid synthesis.

The sterol regulatory-element binding protein (SREBP) family of transcription factors regulates cholesterol, fatty acid, and triglyceride synthesis and metabolism. However, they are also targeted by the ubiquitin ligase Fbw7, a major tumor suppressor, suggesting that they could regulate cell growth. Indeed, enhanced lipid synthesis is a hallmark of many human tumors. Thus, the SREBP pathway has recently emerged as a potential target for cancer therapy. We have previously demonstrated that one of these transcription factors, SREBP1, is stabilized and remains associated with target promoters during mitosis, suggesting that the expression of these target genes could be important as cells enter G1 and transcription is restored. Activation of cyclin D-cdk4/6 complexes is critical for the phosphorylation and inactivation of the retinoblastoma protein (Rb) family of transcriptional repressors and progression through the G1 phase of the cell cycle. Importantly, the cyclin D-cdk4/6-Rb regulatory axis is frequently dysregulated in human cancer. In the current manuscript, we demonstrate that SREBP1 activates the expression of cyclin D1, a coactivator of cdk4 and cdk6, by binding to an E-box in the cyclin D1 promoter. Consequently, inactivation of SREBP1 in human liver and breast cancer cell lines reduces the expression of cyclin D1 and attenuates Rb phosphorylation. Rb phosphorylation in these cells can be rescued by restoring cyclin D1 expression. On the other hand, expression of active SREBP1 induced the expression of cyclin D1 and increased the phosphorylation of Rb in a manner dependent on cyclin D1 and cdk4/6 activity. Inactivation of SREBP1 resulted in reduced expression of cyclin D1, attenuated phosphorylation of Rb, and reduced proliferation. Inactivation of SREBP1 also reduced the insulin-dependent regulation of the cyclin D1 gene. At the same time, SREBP1 is known to play an important role in supporting lipid synthesis in cancer cells. Thus, we propose that the SREBP1-dependent regulation of cyclin D1 coordinates cell proliferation with the enhanced lipid synthesis required to support cell growth.

SMYD3 Promotes Cell Cycle Progression by Inducing Cyclin D3 Transcription and Stabilizing the Cyclin D1 Protein in Medulloblastoma.

Simple SummaryMedulloblastoma is the most common malignant pediatric brain tumor and is classified into four molecular subgroups: Wnt, Shh, Group 3, and Group 4. Of these subgroups, patients with Myc+ Group 3 MB have the worst prognosis. Using an RNAi functional genomic screen, we identified the lysine methyltransferase SMYD3 as a crucial epigenetic regulator responsible for promoting Group 3 MB cell growth. We demonstrated that SMYD3 drives MB cell cycle progression by inducing cyclin D3 transcription and preventing cyclin D1 ubiquitination. Using in vitro and ex vivo studies, we showed that SMYD3 suppression by shRNA and BCI-121 significantly impaired proliferation, resulting in the downregulation of cyclin D3, cyclin D1, and pRBSer795. Moreover, we are the first to show that SMYD3 methylates the cyclin D1 protein, indicating that the SMYD3 stabilizes cyclin D1 through post-translational modification. Collectively, our studies position SMYD3 as a promising treatment option for Group 3 Myc+ MB patients.Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Maximum safe resection, postoperative craniospinal irradiation, and chemotherapy are the standard of care for MB patients. MB is classified into four subgroups: Shh, Wnt, Group 3, and Group 4. Of these subgroups, patients with Myc+ Group 3 MB have the worst prognosis, necessitating alternative therapies. There is increasing interest in targeting epigenetic modifiers for treating pediatric cancers, including MB. Using an RNAi functional genomic screen, we identified the lysine methyltransferase SMYD3, as a crucial epigenetic regulator that drives the growth of Group 3 Myc+ MB cells. We demonstrated that SMYD3 directly binds to the cyclin D3 promoter to activate its transcription. Further, SMYD3 depletion significantly reduced MB cell proliferation and led to the downregulation of cyclin D3, cyclin D1, pRBSer795, with concomitant upregulations in RB in vitro. Similar results were obtained following pharmacological inhibition of SMYD3 using BCI-121 ex vivo. SMYD3 knockdown also promoted cyclin D1 ubiquitination, indicating that SMYD3 plays a vital role in stabilizing the cyclin D1 protein. Collectively, our studies demonstrate that SMYD3 drives cell cycle progression in Group 3 Myc+ MB cells and that targeting SMYD3 has the potential to improve clinical outcomes for high-risk patients.

Di-(2-ethylhexyl) phthalate triggers DNA methyltransferase 1 expression resulting in elevated CpG-methylation and enrichment of MECP2 in the p21 promoter in vitro

Leaching of the plastic constituents leading to their chronic exposure to humans is a major concern for our environmental and occupational health. Our previous and other numerous studies have demonstrated that environmental chemicals like di (2-Ethylhexyl)-phthalate (DEHP) could pose a risk towards the epigenetic mechanisms. Yet, the mechanisms underlying its possible epigenotoxicity are poorly understood. We aimed to assess the impact of DEHP exposure to the human breast cancer cells (MCF-7) and resultant changes in DNA methylation regulators ultimately altering the expression of the cell cycle regulator p21 as a model gene. The MCF-7 cells were exposed to environmentally relevant concentrations (50–500 nM) for 24 h. The results showed that DEHP was proliferative towards the MCF-7 cells while it induced global DNA hypermethylation with selective upregulation of DNMT1 and MECP2. In addition, DEHP significantly reduced p53 protein and its enrichment to the DNMT1 promoter binding site, while elevating SP1 and E2F1 transcription factor levels, stimulating their binding to the promoter DNA. Coincidently, increased DNMT1 level was highly associated with loss of p21 expression and increased cyclin D1 levels. Importantly, the p21, but not cyclin D1 promoter CpG-dinucleotides were hypermethylated after exposure to 500 nM DEHP for 24 h. Furthermore, it was observed that DEHP significantly enriched DNMT1 and MECP2 to the p21 promoter to induce DNA methylation-based epigenetic silencing of p21, resulting in increased cell proliferation. Our results suggest DEHP could potentially induce the epigenetic alterations that might increase the risk of breast cancer, given that the underlying mechanisms should be fully elucidated.

Beta-Caryophyllene Exhibits Anti-Proliferative Effects through Apoptosis Induction and Cell Cycle Modulation in Multiple Myeloma Cells

Simple SummaryMultiple myeloma (MM) is a malignant B-cell neoplasm characterized by the uncontrolled proliferation of plasma cells. MM cells highly express cannabinoid type 2 receptors (CB2Rs), and previous studies have already demonstrated that the Cannabis plant and its derivatives may have anti-emetic as well as anti-neoplastic effects. In the present study, β-caryophyllene (BCP), a natural CB2R agonist, was evaluated for its anti-proliferative and anti-cancer effects. BCP was able to induce the apoptotic mechanism by activating the molecules involved in triggering apoptosis, such as Bax and caspase 3, and it reduced the anti-apoptotic protein Bcl-2; BCP also regulated cell proliferation through sophisticated crosstalk between Akt, β-catenin, and cyclin D1/CDK 4-6 in a concentration-dependent manner. These effects were counteracted by AM630, a CB2R antagonist, thus showing that BCP acts through CB2R. The data obtained so far demonstrate that BCP, thanks to its anti-proliferative effects, might represent an interesting additional therapeutic approach to improve anti-myeloma therapy. Cannabinoid receptors, which are widely distributed in the body, have been considered as possible pharmacological targets for the management of several tumors. Cannabinoid type 2 receptors (CB2Rs) belong to the G protein-coupled receptor family and are mainly expressed in hematopoietic and immune cells, such as B-cells, T-cells, and macrophages; thus, CB2R activation might be useful for treating cancers affecting plasma cells, such as multiple myeloma (MM). Previous studies have shown that CB2R stimulation may have anti-proliferative effects; therefore, the purpose of the present study was to explore the antitumor effect of beta-caryophyllene (BCP), a CB2R agonist, in an in vitro model of MM. Dexamethasone-resistant (MM.1R) and sensitive (MM.1S) human multiple myeloma cell lines were used in this study. Cells were treated with different concentrations of BCP for 24 h, and a group of cells was pre-incubated with AM630, a specific CB2R antagonist. BCP treatment reduced cell proliferation through CB2R stimulation; notably, BCP considerably increased the pro-apoptotic protein Bax and decreased the anti-apoptotic molecule Bcl-2. Furthermore, an increase in caspase 3 protein levels was detected following BCP incubation, thus demonstrating its anti-proliferative effect through apoptosis activation. In addition, BCP regulated AKT, Wnt1, and beta-catenin expression, showing that CB2R stimulation may decrease cancer cell proliferation by modulating Wnt/β-catenin signaling. These effects were counteracted by AM630 co-incubation, thus confirming that BCP’s mechanism of action is mainly related to CB2R modulation. A decrease in β-catenin regulated the impaired cell cycle and especially promoted cyclin D1 and CDK 4/6 reduction. Taken together, these data revealed that BCP might have significant and effective anti-cancer and anti-proliferative effects in MM cells by activating apoptosis, modulating different molecular pathways, and downregulating the cell cycle.

NAP1L1 interacts with hepatoma-derived growth factor to recruit c-Jun inducing breast cancer growth

BackgroundBreast cancer is a common cancer among women in the world. However, its pathogenesis is still to be determined. The role and molecular mechanism of Nucleosome Assembly Protein 1 Like 1 (NAP1L1) in breast cancer have not been reported. Elucidation of molecular mechanism might provide a novel therapeutic target for breast cancer treatment.MethodsA bioinformatics analysis was conducted to determine the differential expression of NAP1L1 in breast cancer and find the potential biomarker that interacts with NAP1L1 and hepatoma-derived growth factor (HDGF). The expression of NAP1L1 in tissues was detected by using immunohistochemistry. Breast cancer cells were transfected with the corresponding lentiviral particles and siRNA. The efficiency of transfection was measured by RT-qPCR and western blotting. Then, MTT, Edu, plate clone formation, and subcutaneous tumorigenesis in nude mice were used to detect the cell proliferation in breast cancer. Furthermore, coimmunoprecipitation (Co-IP) assay and confocal microscopy were performed to explore the detailed molecular mechanism of NAP1L1 in breast cancer.ResultsIn this study, NAP1L1 protein was upregulated based on the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. Consistent with the prediction, immunohistochemistry staining showed that NAP1L1 protein expression was significantly increased in breast cancer tissues. Its elevated expression was an unfavorable factor for breast cancer clinical progression and poor prognosis. Stably or transiently knocking down NAP1L1 reduced the cell growth in vivo and in vitro via repressing the cell cycle signal in breast cancer. Furthermore, the molecular basis of NAP1L1-induced cell cycle signal was further studied. NAP1L1 interacted with the HDGF, an oncogenic factor for tumors, and the latter subsequently recruited the key oncogenic transcription factor c-Jun, which finally induced the expression of cell cycle promoter Cyclin D1(CCND1) and thus the cell growth of breast cancer.ConclusionsOur data demonstrated that NAP1L1 functions as a potential oncogene via interacting with HDGF to recruit c-Jun in breast cancer.

Fukutin Protein Participates in Cell Proliferation by Enhancing Cyclin D1 Expression through Binding to the Transcription Factor Activator Protein-1: An In Vitro Study.

The causative gene of Fukuyama congenital muscular dystrophy (fukutin) is involved in formation of the basement membrane through glycosylation of alpha-dystroglycan. However, there are other proposed functions that have not been fully understood. Using cultured astrocytes (1321N1), we found nuclear localization of fukutin and a positive relationship between fukutin expression and cell proliferation. Among potential proteins regulating cell proliferation, we focused on cyclin D1, by reverse-transcription polymerase chain reaction, Western blotting, immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), and sandwich ELISA. Expression of cyclin D1 was significantly downregulated by fukutin knockdown and significantly upregulated by fukutin overexpression. Moreover, fukutin was proven to bind to the activator protein-1 (AP-1) binding site of cyclin D1 promoter, as well as the AP-1 component c-Jun. The c-Jun phosphorylation status was not significantly influenced by knockdown or overexpression of fukutin. The present results provide in vitro evidence for a novel function of fukutin, which participates in cell proliferation by enhancing cyclin D1 expression through forming a complex with AP-1. It is likely that fukutin is a potential cofactor of AP-1.

Cell Proliferation and Tumor Induction by Ochratoxin A in Mouse Skin and Evaluation of Cyclin D1 and Cyclooxygenase-2 Expressions.

Motivation. Skin tumor is one of the frequent occurring forms of cancer where 2-3 million instances are reported worldwide. The ultraviolet rays along with the environmental pollutants and other contaminants can be the potential factors of skin cancer. Cyclin D1 is a serious gene included in controlling the development through the G1 phase of the cell cycle. Ochratoxin A (OTA) is a naturally existing mycotoxin which majorly occurs in food like grains. It is responsible for producing the splitting of single-strand DNA and is identified to be cancer-causing. It is established as a critical risk factor towards reproductive health in both males and females. Methodology. A single dose of ochratoxin A was used for topical application for assessment of skin tumor promotion activity, hyperplasia, ornithine decarboxylase activity, and expression of cyclin D1 and COX-2 in mouse skin. Enhancement in the synthesis of DNA, activation of the epidermal growth factor receptor, and overexpression of cyclin D1 and COX-2 were noted. Primary murine keratinocyte cell culture was cultured with Waymouth's medium. Western blot analysis and real-time polymerase chain reaction (RT-PCR) were used to detect the expression of cyclin D1 and COX-2. Chromatin immunoprecipitation (ChIP) assays were used to the association between AP-1 transcription and nuclear factor-kappaB (NF-κB) with COX-2 and cyclin D1 promoters. Results. The results found that cyclin D1 and COX-2 were responsible for stimulating OTA-induced PMK proliferation and hyperplasia. Implications. EGFR-mediated pathways were also responsible for tumor promotion due to OTA.

STAT3 activation in thymic epithelial tumors: correlation with cyclin D1, JAK3, and clinical behavior.

Thymic epithelial tumors are the most common adult mediastinal tumors; however, their growth mechanism remains relatively unknown. Among the JAK/STAT pathway-related proteins, which control various intracellular events, STAT3 is deeply involved in cell proliferation. Constitutive activation of STAT3 and the resulting overexpression of cyclin D1 have been confirmed in various tumors, but have not been thoroughly investigated in thymic epithelial tumors. In this study, we immunohistochemically examined STAT3 activation, cyclin D1 expression, and JAK3 activation in thymic epithelial tumors and statistically analyzed their correlation with clinicopathological features. Formalin-fixed paraffin-embedded specimens of 94 thymic epithelial tumors surgically resected at Kyorin University Hospital between 2005 and 2018 were included in this study. pSTAT3, cyclin D1, and pJAK3 were immunohistochemically examined, and the correlation with histology, Masaoka stage, and survival time was statistically analyzed. Cyclin D1 was found to be significantly overexpressed in the STAT3-activated group. This phenomenon was associated with histology and Masaoka stage. JAK3 was also activated in thymic epithelial tumors; however, JAK3 and STAT3 activation were not always correlated. Using survival time analysis, the STAT3-activated group, cyclin D1-expressed group, and JAK3-activated group had significantly lower progression-free survival times than those for both the non-activated and non-expressed groups. STAT3 activation may promote cyclin D1 overexpression in thymic epithelial tumors, and intracellular signaling pathways other than JAK3 may be involved in STAT3 activation. STAT3 activation, cyclin D1 overexpression, and JAK3 activation are biomarker candidates that indicate clinically poor prognosis.

MiR-660 promotes liver cancer cell proliferation by targeting PPP2R2A.

Liver cancer (LC) is the leading cause for tumor-related death worldwide, and microRNAs (miRs) have been demonstrated to regulate the progression of LC. In the current study, the function of miR-660 in LC cells was investigated, and the results indicated that miR-660 was highly expressed in LC tissues and cells. This increased expression promoted LC cell proliferation and increased the percentage of S phase cells, while miR-660 knockdown inhibited cell proliferation and increased the percentage of G0/G1 phase cells. A Ser/Thr phosphatase protein phosphatase 2 regulatory subunit βα (PPP2R2A) was indicated as the target of miR-660, and miR-660 could inhibit PPP2R2A levels. The luciferase reporter assay suggested that miR-660 directly bound to the 3'-untranslated region of PPP2R2A. Additionally, it was revealed that miR-660 inhibited p21 expression and promoted cyclin D1 expression, confirming that miR-660 regulated LC cell proliferation by regulating cell cycle progression. The double knockdown of miR-660 and PPP2R2A promoted LC cell proliferation, suggesting that miR-660 promoted LC proliferation by targeting PPP2R2A.

Sublytic C5b-9 induces glomerular mesangial cell proliferation via ERK1/2-dependent SOX9 phosphorylation and acetylation by enhancing Cyclin D1 in rat Thy-1 nephritis

Glomerular mesangial cell (GMC) proliferation is a histopathological alteration in human mesangioproliferative glomerulonephritis (MsPGN) or in animal models of MsPGN, e.g., the rat Thy‐1 nephritis (Thy-1N) model. Although sublytic C5b-9 assembly on the GMC membrane can trigger cell proliferation, the mechanisms are still undefined. We found that sublytic C5b-9-induced rat GMC proliferation was driven by extracellular signal‐regulated kinase 1/2 (ERK1/2), sry-related HMG-box 9 (SOX9), and Cyclin D1. Here, ERK1/2 phosphorylation was a result of the calcium influx-PKC-α-Raf-MEK1/2 axis activated by sublytic C5b-9, and Cyclin D1 gene transcription was enhanced by ERK1/2-dependent SOX9 binding to the Cyclin D1 promoter (−582 to −238 nt). In addition, ERK1/2 not only interacted with SOX9 in the cell nucleus to mediate its phosphorylation at serine residues 64 (a new site identified by mass spectrometry) and 181 (a known site), but also indirectly induced SOX9 acetylation by elevating the expression of general control non-repressed protein 5 (GCN5), which together resulted in Cyclin D1 synthesis and GMC proliferation. Moreover, our in vivo experiments confirmed that silencing these genes ameliorated the lesions of Thy‐1N rats and reduced SOX9 phosphorylation, acetylation and Cyclin D1 expression. Furthermore, the renal tissue sections of MsPGN patients also showed higher phosphorylation or expression of ERK1/2, SOX9, and Cyclin D1. In summary, these findings suggest that sublytic C5b-9-induced GMC proliferation in rat Thy-1N requires SOX9 phosphorylation and acetylation via enhanced Cyclin D1 gene transcription, which may provide a new insight into human MsPGN pathogenesis.

Role of A-kinase anchoring proteins in cyclic-AMP-mediated Schwann cell proliferation

Proliferation of Schwann cells during peripheral nerve development is stimulated by the heregulin/neuregulin family of growth factors expressed by neurons. However, for neonatal rat Schwann cells growing in culture, heregulins produce only a weak mitogenic response. Supplementing heregulin with forskolin, an agent that elevates cyclic AMP levels, produces a dramatic increase in the proliferation of cultured Schwann cells. The mechanisms underlying this synergistic effect required for Schwann cell proliferation in vivo is not well established. Characterizing the A-kinase anchoring proteins (AKAPs) in Schwann cells might help identify substrates tethered to and phosphorylated by the cAMP-dependent protein kinase A (PKA). Using an RII overlay assay that detects AKAPs that are bound to the type II regulatory subunits of PKA, we identified AKAP150 in Schwann cells. Western blot analysis revealed that additional AKAPs, specifically AKAP95, and yotiao were also present. Disruption of PKA/AKAP interaction with Ht-31 peptide resulted in an increase in luciferase-conjugated cyclin D3 promoter activity. Transfection with sequence-specific AKAP siRNAs for AKAP150 and AKAP95 produced a marked reduction in cell proliferation. Immunoblot analysis revealed that knock down of AKAP95 protein caused a significant decrease in expression of the cell cycle regulatory proteins cyclin D2, cyclin D3 and the cell survival signal Akt/Protein Kinase B (Akt/PKB). Morphological characterization of Schwann cell AKAPs indicated the presence of nuclear (AKAP95), cytoplasm-associated (AKAP150) and perinuclear (yotiao) A-kinase anchoring proteins. These results indicate a role for AKAP95 and AKAP150 in the synergistic response of Schwann cells to treatment with heregulin and forskolin.

Circular RNA circGLIS3 promotes bladder cancer proliferation via the miR-1273f/SKP1/Cyclin D1 axis

Extensive research confirmed that circRNA can play a regulatory role in various stages of tumors by interacting with various molecules. Identifying the differentially expressed circRNA in bladder cancer and exploring its regulatory mechanism on bladder cancer progression are urgent. In this study, we screened out a circRNA-circGLIS3 with a significant upregulation trend in both bladder cancer tissues and cells. Bioinformatics prediction results showed that circGLIS3 may be involved in multiple tumor-related pathways. Function gain and loss experiments verified circGLIS3 can affect the proliferation, migration, and invasion of bladder cancer cells in vitro. Moreover, silencing circGLIS3 inhibited bladder cancer cell growth in vivo. Subsequent research results indicated circGLIS3 regulated the expression of cyclin D1, a cell cycle–related protein, and cell cycle progression. Mechanically, circGLIS3 upregulates the expression of SKP1 by adsorbing miR-1273f and then promotes cyclin D1 expression, ultimately promoting the proliferation of bladder cancer cells. In summary, our study indicates that circGLIS3 plays an oncogene role in the development of bladder cancer and has potential to be a candidate for bladder cancer.Graphical abstract

GDF-5 promotes epidermal stem cells proliferation via Foxg1-cyclin D1 signaling

ObjectiveEpidermal stem cells (EpSCs) can self-renew, which are responsible for the long-term maintenance of the skin, and it also plays a critical role in wound re-epithelization, but the mechanism underlying EpSCs proliferation is unclear. GDF-5, also known as BMP-14, is a member of the BMP family and can be used as a self-renewal supporter. Here, we studied the effects of GDF-5 on mouse EpSCs proliferation mechanism in wound healing.MethodsFirstly, the effects of GDF-5 on EpSCs proliferation was tested by using CCK8 reagent and PCNA expression was analyzed by Western blotting. Secondly, we screened genes that promote EpSCs proliferation in the FOX and cyclin family by qPCR, and then the protein expression level of the selected genes was further analyzed by Western blotting. Thirdly, siRNA plasmids and pAdEasy adenovirus were transfected or infected, respectively, into mouse EpSCs to detect the effect of target genes on GDF-5-induced cell proliferation. Furthermore, we injected GDF-5 to a deep partial thickness burn mouse model for finding out whether EpSCs proliferation can be detected by immunohistochemical. Finally, the relevant target genes were analyzed by qPCR, immunoblotting, and dual-luciferase reporter gene detection.ResultsWe discovered that 100 ng/ml recombinant mouse GDF-5 was the optimal concentration for promoting mouse EpSCs proliferation. Through preliminary screened by qPCR, we found that Foxg1 and cyclin D1 could be the downstream molecules of GDF-5, and the results were confirmed by Western blotting. And the effect of GDF-5 on mouse EpSCs proliferation was adjusted by Foxg1/cyclin D1 in vitro and in vivo. Besides, GDF-5-induced transcription of cyclin D1 was regulated by Foxg1-mediated cyclin D1 promoter activity.ConclusionThis paper showed that GDF-5 promotes mouse EpSCs proliferation via Foxg1-cyclin D1 signal pathway. It is suggested that GDF-5 may be a new approach to make EpSCs proliferation which can be used in wound healing.

KDM6B is an androgen regulated gene and plays oncogenic roles by demethylating H3K27me3 at cyclin D1 promoter in prostate cancer

Lysine (K)-specific demethylase 6B (KDM6B), a stress-inducible H3K27me3 demethylase, plays oncogenic or antitumoral roles in malignant tumors depending on the type of tumor cell. However, how this histone modifier affects the progression of prostate cancer (PCa) is still unknown. Here we analyzed sequenced gene expression data and tissue microarray to explore the expression features and prognostic value of KDM6B in PCa. Further, we performed in vitro cell biological experiments and in vivo nude mouse models to reveal the biological function, upstream and downstream regulation mechanism of KDM6B. In addition, we investigated the effects of a KDM6B inhibitor, GSK-J4, on PCa cells. We showed that KDM6B overexpression was observed in PCa, and elevated KDM6B expression was associated with high Gleason Score, low serum prostate-specific antigen level and shorted recurrence-free survival. Moreover, KDM6B prompted proliferation, migration, invasion and cell cycle progression and suppressed apoptosis in PCa cells. GSK-J4 administration could significantly suppress the biological function of KDM6B in PCa cells. KDM6B is involved in the development of castration-resistant prostate cancer (CRPC), and combination of MDV3100 plus GSK-J4 is effective for CRPC and MDV3100-resistant CRPC. Mechanism exploration revealed that androgen receptor can decrease the transcription of KDM6B and that KDM6B demethylates H3K27me3 at the cyclin D1 promoter and cooperates with smad2/3 to prompt the expression of cyclin D1. In conclusion, our study demonstrates that KDM6B is an androgen receptor regulated gene and plays oncogenic roles by promoting cyclin D1 transcription in PCa and GSK-J4 has the potential to be a promising agent for the treatment of PCa.