Cyclin D1 Transcription Research Articles

In silico identification and in vitro validation of alpha-hederin as a potent inhibitor of Wnt/β-catenin signaling pathway in breast cancer stem cells.

Cancer stem cells (CSCs) play a vital role in metastasis, recurrence and chemoresistance in breast cancer. β-catenin, which is a frequently over activated protein in CSCs, binds to T-cell factor/lymphoid enhancer factor (Tcf/Lef) family transcription factors leading to ectopic expression of Wnt pathway responsive genes necessary for the maintenance and action of CSCs. With the aim of identifying a small molecules that can effectively eliminate CSCs, molecular docking studies were performed against the Tcf/Lef binding hotspot on β-catenin using a library of 100 natural or synthetic small molecules. Small molecule ligands giving docking energy better than -7kcal/mol were further investigated by binding interactions analysis and molecular dynamics (MD) simulations. These compounds were then investigated in vitro, for cytotoxicity against CSCs isolated from MDA-MB-231 triple negative breast cancer cells. Alpha-hederin (AH) was identified as the only compound in the selected library that has cytotoxicity against breast CSCs. AH was further investigated for it's ability to regulate Wnt pathway target genes (Cyclin D1 and CD44)and the tumor suppressor p53by real-time quantitative PCR. Absorption, distribution, metabolism, excretion and toxicity properties of the AH was predicted in silico. AH significantly down regulated the transcription of Cyclin D1 and CD44 while up-regulating the transcription of p53. AH was predicted to have acceptable drug likeness. Although AH is currently known to inhibit the growth of various cancer cells in vitro, present study demonstrated for the first time that it is a potent inhibitor of Wnt/β-catenin signaling pathway and induce apoptosis in breast CSCs.

PC4: A new regulator of cyclin D1 transcript levels.

The expression of cyclin proteins is tightly regulated during the cell cycle, to allow precise activation of cyclin-dependent kinases. In this issue, Pan et al. (https://doi.org/10.1083/jcb.202308066) identify an RNA-binding protein, PC4, as a regulator of cyclin D1 mRNA stability in hepatocellular carcinoma cells. This study provides a new mechanism regulating the levels of a key cell cycle protein, cyclin D1, in human cells.

Targeted Disruption of PDCD2 Delays G1/S Transition in Lung Carcinoma by Inhibiting Cyclin D1 Transcription

We previously reported on high expression of define PDCD2 in human malignancies. Knockdown of PDCD2 reduced the proliferation of leukemia and lung carcinoma cells. However, the mechanism by which PDCD2 reduces tumor proliferation remains unclear. This study tested the hypothesis that lowered PDCD2 would delay the proliferation of A549 lung carcinoma cells. Entry of A549 cells into S-phase was significantly (p-value 0.05) delayed when PDCD2 was knockdown. This correlated with a significant downregulation of cyclin D1, and AKT phosphorylation. Inhibition of the PI3K/AKT signaling pathway by Ly294002 decreased levels of PDCD2. These findings are consistent with a role for PDCD2 to mediate the entry of A549 cells into the cell cycle. Resting A549 cells showed PDCD2 localizing in the nucleus and plasma membrane and became diffuse with cell division, suggesting that PDCD2 is mitogen-dependent and may be involved in the proper timing of cell cycle. These findings may represent a promising venue to develop PDCD2 in clinical applications.

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

Experimental study on the therapeutic effect and mechanism of erlotinib on non-proliferative diabetic retinopathy

LAIR1 drives glioma progression by nuclear focal adhesion kinase dependent expressions of cyclin D1 and immunosuppressive chemokines/cytokines

Leukocyte-associated immunoglobulin-like receptor-1 (LAIR1), an immune receptor containing immunoreceptor tyrosine-based inhibiory motifs (ITIMs), has emerged as an attractive target for cancer therapy. However, the intrinsic function of LAIR1 in gliomas remains unclear. In this study, the poor prognosis of glioma patients and the malignant proliferation of glioma cells in vitro and in vivo were found to be closely correlated with LAIR1. LAIR1 facilitates focal adhesion kinase (FAK) nuclear localization, resulting in increased transcription of cyclin D1 and chemokines/cytokines (CCL5, TGFβ2, and IL33). LAIR1 specifically supports in the immunosuppressive glioma microenvironment via CCL5-mediated microglia/macrophage polarization. SHP2Q510E (PTP domain mutant) or FAKNLM (non-nuclear localizing mutant) significantly reversed the LAIR1-induced growth enhancement in glioma cells. In addition, LAIR1Y251/281F (ITIMs mutant) and SHP2Q510E mutants significantly reduced FAK nuclear localization, as well as CCL5 and cyclin D1 expression. Further experiments revealed that the ITIMs of LAIR1 recruited SH2-containing phosphatase 2 (SHP2), which then interacted with FAK and induced FAK nuclear localization. This study uncovered a critical role for intrinsic LAIR1 in facilitating glioma malignant progression and demonstrated a requirement for LAIR1 and SHP2 to enhance FAK nuclear localization.

αKG-driven RNA polymerase II transcription of cyclin D1 licenses malic enzyme 2 to promote cell-cycle progression

Increased metabolic activity usually provides energy and nutrients for biomass synthesis and is indispensable for the progression of the cell cycle. Here, we find a role for α-ketoglutarate (αKG) generation in regulating cell-cycle gene transcription. A reduction in cellular αKG levels triggered by malic enzyme 2 (ME2) or isocitrate dehydrogenase 1 (IDH1) depletion leads to a pronounced arrest in G1 phase, while αKG supplementation promotes cell-cycle progression. Mechanistically, αKG directly binds to RNA polymerase II (RNAPII) and increases the level of RNAPII binding to the cyclin D1 gene promoter via promoting pre-initiation complex (PIC) assembly, consequently enhancing cyclin D1 transcription. Notably, αKG addition is sufficient to restore cyclin D1 expression in ME2- or IDH1-depleted cells, facilitating cell-cycle progression and proliferation in these cells. Therefore, our findings indicate a function of αKG in gene transcriptional regulation and cell-cycle control.

GDNF-RET signaling and EGR1 form a positive feedback loop that promotes tamoxifen resistance via cyclin D1

BackgroundRearranged during transfection (RET) tyrosine kinase signaling has been previously implicated in endocrine resistant breast cancer, however the mechanism by which this signaling cascade promotes resistance is currently not well described. We recently reported that glial cell-derived neurotrophic factor (GDNF)-RET signaling appears to promote a positive feedback loop with the transcription factor early growth response 1 (EGR1). Here we investigate the mechanism behind this feedback loop and test the hypothesis that GDNF-RET signaling forms a regulatory loop with EGR1 to upregulate cyclin D1 (CCND1) transcription, leading to cell cycle progression and tamoxifen resistance.MethodsTo gain a better understanding of the GDNF-RET-EGR1 resistance mechanism, we studied the GDNF-EGR1 positive feedback loop and the role of GDNF and EGR1 in endocrine resistance by modulating their transcription levels using CRISPR-dCAS9 in tamoxifen sensitive (TamS) and tamoxifen resistant (TamR) MCF-7 cells. Additionally, we performed kinetic studies using recombinant GDNF (rGDNF) treatment of TamS cells. Finally, we performed cell proliferation assays using rGDNF, tamoxifen (TAM), and Palbociclib treatments in TamS cells. Statistical significance for qPCR and chromatin immunoprecipitation (ChIP)-qPCR experiments were determined using a student’s paired t-test and statistical significance for the cell viability assay was a one-way ANOVA.ResultsGDNF-RET signaling formed a positive feedback loop with EGR1 and also downregulated estrogen receptor 1 (ESR1) transcription. Upregulation of GDNF and EGR1 promoted tamoxifen resistance in TamS cells and downregulation of GDNF promoted tamoxifen sensitivity in TamR cells. Additionally, we show that rGDNF treatment activated GDNF-RET signaling in TamS cells, leading to recruitment of phospho-ELK-1 to the EGR1 promoter, upregulation of EGR1 mRNA and protein, binding of EGR1 to the GDNF and CCND1 promoters, increased GDNF protein expression, and subsequent upregulation of CCND1 mRNA levels. We also show that inhibition of cyclin D1 with Palbociclib, in the presence of rGDNF, decreases cell proliferation and resensitizes cells to TAM.ConclusionOutcomes from these studies support the hypotheses that GDNF-RET signaling forms a positive feedback loop with the transcription factor EGR1, and that GDNF-RET-EGR1 signaling promotes endocrine resistance via signaling to cyclin D1. Inhibition of components of this signaling pathway could lead to therapeutic insights into the treatment of endocrine resistant breast cancer.

Chemotherapy-Induced Senescence Reprogramming Promotes Nasopharyngeal Carcinoma Metastasis by circRNA-Mediated PKR Activation.

Senescence is associated with tumor metastasis and chemotherapy resistance, yet the mechanisms remain elusive. Here, it is identified that nasopharyngeal carcinoma (NPC) patients who developed distant metastasis are characterized by senescence phenotypes, in which circWDR37 is a key regulator. CircWDR37 deficiency limits cisplatin or gemcitabine-induced senescent NPC cells from proliferation, migration, and invasion. Mechanistically, circWDR37 binds to and dimerizes double-stranded RNA-activated protein kinase R (PKR) to initiate PKR autophosphorylation and activation. Independent of its kinase activity, phosphorylated PKR induces I-kappaB kinase beta (IKKβ) phosphorylation, binds to and releases RELA from NF-κB inhibitor alpha (IκBα) to trigger nuclear factor kappa B (NF-κB) activation, thereby stimulating cyclin D1 (CCND1)and senescence-associated secretory phenotype component gene transcription in a circWDR37-dependent manner. Low circWDR37 levels correlate with chemotherapy response and favorable survival in NPC patients treated with gemcitabine or cisplatin induction chemotherapy. This study uncovers a new mechanism of circWDR37 activated PKR in senescence-driven metastasis and provides appealing therapeutic targets in NPC.

Simulated microgravity altered the cell cycle progression of porcine granulosa cells

Microgravity has been shown to markedly affect reproduction in humans and animals, especially reproductive organs such as the ovaries. Granulosa cells are one of the important components of the ovary, playing an important role in supporting oocyte maturation and fertilization. However, the effects of microgravity on granulosa cells have not been well characterized. This study aimed to assess the effects of simulated microgravity (SMG) on the cell cycle progression of porcine granulosa cells (pGCs). The pGCs were induced SMG for 72 h by Gravite® simulator, while cells of the control group were treated in normal conditions. Cell cycle analysis revealed that SMG condition induced an increase of the ratio of pGCs in the G0/G1 phase, leading to the cell cycle arrest phase, while the ratio of pGCs in the G2/M phase was decreased. There was no difference in the cell ratio of the S phase between the control group and the SMG group. Real-time RT-PCR analysis indicated that the expression of cdk4 and cdk6 transcripts of pGCs from the SMG group was lower than the control group. This down-regulation was also observed cyclin A and cyclin D1 transcript expression in pGCs from the SMG group. Immunostaining displayed the lower exhibition of microfilament and microtubule in pGCs from the SMG group comparing to the control group. Western blot analysis showed that the expression of β-actin and α-tubulin was reduced in pGCs from the SMG group. These changes contributed to the alteration of cytoskeletal structure, including microfilaments and microtubules, which affect cell division. These results revealed that the SMG condition induced changes in the cell cycle progression of pGCs.

Abstract A23: Alternative cyclin D1 transcripts in Mantle Cell Lymphoma

Abstract Mantle Cell Lymphoma (MCL) is a rare aggressive subtype of Non-Hodgkin’s Lymphoma. Upon diagnosis, treatments are varied and there is no universal standard of care. MCL is considered clinically incurable upon relapse. Better understanding of the development of MCL at the molecular level is needed. The initiating event is a hallmark (11; 14) translocation event that juxtaposes cyclin D1 (CCND1) downstream of the IgH enhancer element thus activating protein expression. This IgH recombination precedes the MCL diagnosis by several years. By itself, expression of cyclin D1 is insufficient for tumorigenesis. However, increased cyclin D1 expression combined with alterations in one of several DNA repair genes facilitates lymphomagenesis characterized by high genomic instability. Interestingly at the mRNA level, CCND1 transcripts containing the full length 3`untranslated regions (3`UTRs) have a half-life of ~30 minutes. CCND1 transcripts containing truncated 3`UTRs have a longer half-life up to ~8 hours resulting in increased translation. A subset of MCL patients with highly proliferative disease express CCND1 mRNAs with truncated 3`UTRs. Wiestner et al showed that truncated CCND1 3`UTR expression was associated with a reduction in patient survival by almost two years. While CCND1 3`UTR shortening in 20% of patient samples was due to mutations, the mechanisms driving this event in the rest of patients remain unknown. Our goal is to identify the different truncated CCND1 transcripts in MCL and determine how they are expressed. Using 3′Rapid Amplification of cDNA ends (3′RACE) we previously identified a novel fusion transcript where a short CCND1 3′UTR sequence was linked to intronic sequences of the MRCKB/CDC42BPB gene. In order to measure levels of expression of differently sized CCND1 3`UTR transcripts we used specifically designed primers for qRT-PCR. To determine the type of truncated CCND1 3`UTR expressed in other MCL cell lines we used 3′RACE. Iso Sequencing was used to verify the occurrence of the novel CCND1/MRCK fusion transcript. Cell viability assays were used to determine the impact of targeting the CCND1/MRCK fusion or cyclin D1 activated cyclin dependent kinase 4/6 inhibitor. Our qRT-PCR results showed that most of the MCL cell lines predominantly expressed CCND1 transcripts with truncated 3′UTRs with the exception of Mino-1 where the longer 3′UTR was more dominant. The 3′RACE results indicate that different mechanisms may result in the expression of the truncated CCND1 3`UTR transcript in each cell line. Iso Sequencing data identified 1,032 total reads supporting the CCND1/MRCK fusion transcript verifying our 3’RACE results. Our results suggest that there are different molecular mechanisms being used to regulate cyclin D1 expression in MCL. Cell viability assays showed decreased cell proliferation. In conclusion, identifying and understanding the mechanisms of expression of different CCND1 transcripts has potential in understanding MCL pathogenesis and the development of MCL therapeutics. Funding: 1R01-GM135361-01 to C. P. Masamha. Citation Format: Naazneen Khan, Chioniso P Masamha. Alternative cyclin D1 transcripts in Mantle Cell Lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A23.

The effects of Jiawei Duhuo Jisheng mixture on Wnt/β-catenin signaling pathway in the synovium inflamed by knee osteoarthritis: An in vitro and in vivo experiment

Ethnopharmacological relevanceKnee osteoarthritis (KOA) is one of the common age-degenerative diseases. Recent studies have demonstrated that the pathogenesis of KOA is closely related to synovial lesions. Jiawei Duhuo Jisheng mixture (JDJM) has shown great potential in the treatment of KOA. However, the effect and mechanism of JDJM on synovial lesions of KOA remain unclear. Aim of the studyThe regulatory effect of JDJM on the Wnt/β-catenin signaling pathway in KOA inflamed synovium was studied via in vitro and in vivo experiments, respectively. Materials and methodsFor the in vitro experiment, fibroblasts were isolated from the rabbit synovium with KOA. The fibroblasts were grouped as follows: the vehicle group was given 0.5% FBS; the inhibitor group was treated with 0.5% volume fraction of XAV939; the normal serum groups and JDJM serum groups were treated with 5%, 10%, and 20% volume fractions of normal serum and JDJM-containing serum. The expression levels of Wnt3a, β-catenin, Cyclin D1, metalloproteinase-7(MMP-7) and cyclooxygenase-2(COX-2) were detected by different assays 48 and 72 h after the intervention.For the in vivo experiment, the rabbit KOA model was prepared using the improved Hulth modeling method, whereby all rabbits were randomly divided into normal control, model control, positive control, and traditional Chinese medicine (TCM) groups. The expression levels of Wnt3a, β-catenin, Cyclin D1, MMP-7 and COX-2 were detected by different assays in the 2, 4, and 8 weeks of treatment. ResultsIn the two test results of in vitro experiments, the normal serum group was compared with the JDJM-containing serum group with the same volume fraction, demonstrating that mRNA transcription and protein expression levels of Wnt3a, β-catenin, Cyclin D1, MMP-7, and COX-2 in the latter decreased (P < 0.05), with more pronounced effects observed in the group treated with 20% volume fraction of JDJM serum. Compared with the inhibitor group, there was no significant difference (P > 0.05) in the mRNA transcription and protein expression levels, i.e., Wnt3a, β-catenin, Cyclin D1, and MMP-7 were observed in the JDJM serum groups, except for a significant decrease (P < 0.05) in the level of mRNA transcription and protein expression of COX-2.Based on the in vivo experiment, compared to the model control group, articular cartilage, synovial hyperplasia, and the inflammatory reaction of the TCM group at different treatment times were significantly improved. The mRNA transcription level of Wnt3a, β-catenin, Cyclin D1, MMP-7 and COX-2 detected by RT-qPCR and the protein expression level of Wnt3a, β-catenin, Cyclin D1, MMP-7 and COX-2 detected by Western blot were significantly reduced (P < 0.05), and the effect was more evident at the eighth week. ConclusionJDJM can regulate the synovial Wnt/β-catenin signaling pathway in KOA models, reduce the mRNA transcription and protein expression levels of Wnt3a, β-catenin, Cyclin D1, MMP-7, and COX-2 in the synovium, thus inhibiting synovial inflammation and protecting articular cartilage, which could be the key mechanism of action in treating this disease.

RNA Helicase DHX37 Facilitates Liver Cancer Progression by Cooperating with PLRG1 to Drive Superenhancer-Mediated Transcription of Cyclin D1.

This work characterizes a novel mechanism of superenhancer-driven cyclin D1 upregulation by DHX37 and PLRG1, implicating this pathway as a potential therapeutic target in hepatocellular carcinoma.

POS-079 14-3-3ζ：A PROTECTOR IN CISPLATIN-INDUCED ACUTE KIDNEY INJURY

Cisplatin is an anticancer agent marred by acute kidney injury (AKI). Limiting this adverse effect may allow the use of higher doses to improve its efficacy. Basing on our previous study verifying that exosomes from bone marrow mesenchymal stem cells (BMSCs) can promote the repair in cisplatin-induced AKI(AKI), the objective of this study was to identify a candidate that targets to alleviate CKI. We analyzed cisplatin-injured cells and BMSCs-treated cells by using iTRAQ isobaric tags and 2D nano LC-MS/MS.14-3-3 expression in the rat renal tubule epithelial cells (NRK-52E) was evaluated through RT-qPCR and western blotting analyses. 14-3-3ζ overexpression lentiviruses and small interfering RNA (siRNA) transfection were applied to test the its function in CKI. 1. By using the iTRAQ method, we analyzed 3539 proteins, of which 266 showed significantly different expression in BMSCs-treated cells, including 14-3-3. Ingenuity pathway analysis (IPA) showed that the majority of differentially expressed proteins (DEPs) were associated with cell death and survival, cell cycle, cellular movement and development. Further analysis indicated that 14-3-3 had the strongest effect on cell cycle and apoptosis.2. The expression of 14-3-3ζ in NRK-52E was downregulated when administered with cisplatin. Along with this, the cell viability and proliferation level in cisplatin group were dramatically reduced as measured by cck8 and flow cytometry analysis. Besides, the proportion of apoptotic cells increased sharply comparing to control group.3. pro-apoptotic protein cleaved-caspase3 increased and anti-apoptotic protein bcl-2 reduced. Further, Co-IP assays showed the interaction between 14-3-3ζ and β-catenin. Si14-3-3ζ downregulated expression of β-catenin and its nuclear translocation as revealed by western blot and immunofluorescence analysis. Besides, lower β-catenin nuclear translocation induced by knockdown of 14-3-3ζ decreased transcription of cyclin D1.4. Similarly, overexpression of 14-3-3ζ exerted a positively protective effect on NRK-52E cells administered with cisplatin, accompanied by the motivation of β-catenin/cyclinD1 pathway. Together, our findings indicate that 14-3-3ζ interacts with β-catenin, contributing to proliferation and apoptosis through β-catenin/cyclinD1 pathway in CKI, which lead to a previously unrecognized dual function and have potential for treatment of CKI.

22-kD growth hormone-induced nuclear GHR/STAT5/CyclinD1 signaling pathway plays an important role in promoting mesenchymal stem cell proliferation.

Growth hormone (GH) exhibited the important biological activities in the mesenchymal stem cell (MSCs). However, the cellular behavior and properties of GH/GHR in MSCs remain unclear. A series of experiments (such as confocal laser scanning microscope [CLSM] and Western-blot) were performed to systematically investigate the cellular behavior of GH/GHR in MSCs, and the results showed that GH/GHR not only internalized into the cytoplasm, but also transported into the cell nuclei of MSCs. Furthermore, we studied the molecular mechanism by which GH/GHR internalized into cell, and the results indicated that clathrin plays more important role in the process of GHR internalization. More importantly, it can be found that nuclear-targeted GHR has the important biological functions in MSCs, which could promote MSCs proliferation. We further revealed the molecular mechanism by which nuclear-localized GHR regulates MSCs proliferation, and found that nuclear-targeted GHR enhanced the phosphorylation of STAT5, and the activated STAT5 initiates the transcription of CyclinD1, after which, the complex of CyclinD1 and CDK4 further phosphorylates Rb, and the activated Rb releases E2F1, the released E2F1 ultimately realizes the biological function of GH promoting cell proliferation. In short, in the current study，we used MSCs as a model to study the cellular behavior and properties of GH/GHR, and found that GH/GHR can internalize into cell cytoplasm and transport into the cell nuclei. Further work showed that nuclear GHR could drive cell proliferation via GHR/STAT5/CyclinD1 signaling pathway. The current research has laid an important foundation for further study on the regulatory effect of GH on MSCs.

TIP30 overcomes gefitinib resistance by regulating cytoplasmic and nuclear EGFR signaling in non-small-cell lung cancer.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) (eg, gefitinib) exert potent therapeutic efficacy in non–small‐cell lung cancer (NSCLC) harboring EGFR‐activating mutations. However, the resistance to EGFR TKIs limits their clinical therapeutic efficacy. TIP30, a newly identified tumor suppressor, appears to be involved in the regulation of cytoplasmic and nuclear EGFR signaling in NSCLC. Our previous study demonstrated that TIP30 regulated EGF‐dependent cyclin D1 transcription in human lung adenocarcinoma and suppressed tumorigenesis. In the present study, the involvement of TIP30 in combating gefitinib resistance in NSCLC was determined for the first time in vitro and in vivo. Gain and loss of function studies showed that overexpression of TIP30 effectively sensitized cells to gefitinib in vitro, whereas TIP30 inhibition promoted gefitinib cell resistance. Moreover, TIP30 negatively regulated the activation of the p‐AKT and p‐MEK signaling pathways in PC9/GR. Importantly, PC9/GR harbored high levels of nuclear EGFR, and overexpression of TIP30 restored irregular EGFR trafficking and degradation from early endosomes to the late endosomes, decreasing the nuclear accumulation of EGFR, which may partly or totally inhibit EGFR‐mediated induction of c‐Myc transcription. Xenographic tumors induced by overexpression of TIP30 by PC9/GR cells in nude mice were suppressed compared with their original counterparts. Overall, it was revealed that TIP30 overexpression restored gefitinib sensitivity in NSCLC cells and attenuated the cytoplasmic and nuclear EGFR signaling pathways and may be a promising biomarker in gefitinib resistance in NSCLC.

FAK in the nucleus prevents VSMC proliferation by promoting p27 and p21 expression via Skp2 degradation.

Vascular smooth muscle cells (VSMCs) normally exhibit a very low proliferative rate. Vessel injury triggers VSMC proliferation, in part, through focal adhesion kinase (FAK) activation, which increases transcription of cyclin D1, a key activator for cell cycle-dependent kinases (CDKs). At the same time, we also observe that FAK regulates the expression of the CDK inhibitors (CDKIs) p27 and p21. However, the mechanism of how FAK controls CDKIs in cell cycle progression is not fully understood. We found that pharmacological and genetic FAK inhibition increased p27 and p21 by reducing stability of S-phase kinase-associated protein 2 (Skp2), which targets theCDKIs for degradation. FAK N-terminal domain interacts with Skp2 and an APC/C E3 ligase activator fizzy-related 1 (Fzr1) in the nucleus, which promote ubiquitination and degradation of both Skp2 and Fzr1. Notably, overexpression of cyclin D1 alone failed to promote proliferation of genetic FAK kinase-dead (KD) VSMCs, suggesting that the FAK-Skp2-CDKI signalling axis is distinct from the FAK-cyclin D1 pathway. However, overexpression of both cyclin D1 and Skp2 enabled proliferation of FAK-KD VSMCs, implicating that FAK ought to control both activating and inhibitory switches for CDKs. In vivo, wire injury activated FAK in the cytosol, which increased Skp2 and decreased p27 and p21 levels. Both pharmacological FAK and genetic FAK inhibition reduced Skp2 expression in VSMCs upon injury, which significantly reduced intimal hyperplasia through elevated expression of p27 and p21. This study revealed that nuclear FAK-Skp2-CDKI signalling negatively regulates CDK activity in VSMC proliferation.

Smooth Muscle Cell-Specific PKM2 (Pyruvate Kinase Muscle 2) Promotes Smooth Muscle Cell Phenotypic Switching and Neointimal Hyperplasia.

[Figure: see text].

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.

The subcellular redistribution of NLRC5 promotes angiogenesis via interacting with STAT3 in endothelial cells.

Angiogenesis is a critical step in repair of tissue injury. The pattern recognition receptors (PRRs) recognize pathogen and damage associated molecular patterns (DAMPs) during injury and achieve host defense directly. However, the role of NLR family CARD domain containing 5 (NLRC5), an important member of PPRs, beyond host defense in angiogenesis during tissue repair remains unknown.Methods: In vitro, western blot and real-time PCR (RT-PCR) were used to detect the expression of NLRC5 in endothelial cells (ECs). Immunofluorescence microscopy was used to reveal the subcellular location of NLRC5 in ECs. Cell proliferation, wound healing, tube formation assays of ECs were performed to study the role of NLRC5 in angiogenesis. By using Tie2Cre-NLRC5flox/flox mice and bone marrow transplantation studies, we defined an EC-specific role for NLRC5 in angiogenesis. Mechanistically, co-immunoprecipitation studies and RNA sequencing indicated that signal transducer and activator of transcription 3 (STAT3) was the target of NLRC5 in the nucleus. And Co-IP was used to verify the specific domain of NLRC5 binding with STAT3. ChIP assay determined the genes regulated by interaction of STAT3 and NLRC5.Results: Knockdown of NLRC5 in vitro or in vivo inhibited pathological angiogenesis, but had no effect on physiological angiogenesis. NLRC5 was also identified to bind to STAT3 in the nucleus required the integrated death-domain and nucleotide-binding domain (DD+NACHT domain) of NLRC5. And the interaction of STAT3 and NLRC5 could enhance the transcription of angiopoietin-2 (Ang2) and cyclin D1 (CCND1) to participate in angiogenesis.Conclusions: In the ischemic microenvironment, NLRC5 protein accumulates in the nucleus of ECs and enhances STAT3 transcriptional activity for angiogenesis. These findings establish NLRC5 as a novel modulator of VEGFA signaling, providing a new target for angiogenic therapy to foster tissue regeneration.

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.