Canonical Thyroid Hormone Receptor ß Action Stimulates Hepatocyte Proliferation in Male Mice.

CONTEXT: 3,5,3'-L-triiodothyronine (T3) is a potent inducer of hepatocyte proliferation via the Wnt/ß-catenin signaling pathway. Previous studies suggested the involvement of rapid noncanonical thyroid hormone receptor (TR) ß signaling, directly activating hepatic Wnt/ß-catenin signaling independent from TRß DNA binding. However, the mechanism by which T3 increases Wnt/ß-catenin signaling in hepatocytes has not yet been determined. OBJECTIVE: We aimed to determine whether DNA binding of TRß is required for stimulation of hepatocyte proliferation by T3. METHODS: Wild-type (WT) mice, TRß knockout mice (TRß KO), and TRß mutant mice with either specifically abrogated DNA binding (TRß GS) or abrogated direct phosphatidylinositol 3 kinase activation (TRß 147F) were treated with T3 for 6 hours or 7 days. Hepatocyte proliferation was assessed by Kiel-67 (Ki67) staining and apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Activation of ß-catenin signaling was measured in primary murine hepatocytes. Gene expression was analyzed by microarray, gene set enrichment analysis (GSEA), and quantitative reverse transcription polymerase chain reaction. RESULTS: T3 induced hepatocyte proliferation with an increased number of Ki67-positive cells in WT and TRß 147F mice (9.2% ±â€…6.5% and 10.1% ±â€…2.9%, respectively) compared to TRß KO and TRß GS mice (1.2% ±â€…1.1% and 1.5% ±â€…0.9%, respectively). Microarray analysis and GSEA showed that genes of the Wnt/ß-catenin pathway-among them, Fzd8 (frizzled receptor 8) and Ctnnb1 (ß-catenin)-were positively enriched only in T3-treated WT and TRß 147F mice while B-cell translocation gene anti-proliferation factor 2 was repressed. Consequently, expression of Ccnd1 (CyclinD1) was induced. CONCLUSIONS: Instead of directly activating Wnt signaling, T3 and TRß induce key genes of the Wnt/ß-catenin pathway, ultimately stimulating hepatocyte proliferation via CyclinD1. Thus, canonical transcriptional TRß action is necessary for T3-mediated stimulation of hepatocyte proliferation.

CDCA2 triggers in vivo and in vitro proliferation of hepatocellular carcinoma by activating the AKT/CCND1 signaling.

PURPOSE: This study aims to elucidate the biological functions of CDCA2 (cell division cycle associated 2) in hepatocellular carcinoma (HCC) progression and the potential mechanism. METHODS: CDCA2 levels in HCC tissues and cell lines were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between CDCA2 and clinical characteristics in HCC patients was analyzed. Cox proportional-hazards model was applied for assessing the potential factors influencing overall survival in HCC. Three CDCA2 siRNAs were generated and the most effective one was used in the following experiments. After knockdown of CDCA2 in HCC-LM3 cells, clonality and viability were examined. Meanwhile, cell cycle progression was detected by flow cytometry. Relative levels of CDCA2, p21, p27, CDK2, CCND1, CCNE1 and CCNB1 in HCC-LM3 cells were determined by qRT-PCR. The activation of the protein kinase B (Akt) signaling was examined by Western blot. Subsequently, we constructed HCC xenograft model in nude mice. Tumor volume and tumor weight of xenografted HCC were recorded. RESULTS: CDCA2 was upregulated in HCC tissues than that of para-tumor ones, especially HCC tissues with larger than 5 cm in tumor size or vascular invasion. CDCA2 level was related to tumor size, vascular invasion and tumor differentiation in HCC. Knockdown of CDCA2 inhibited clonality and viability in HCC-LM3 cells, and arrested cell cycle progression in G1 phase via downregulating CCND1. The phosphatidilinositol 3-kinase (PI3K)/Akt was activated by CDCA2 during the progression of HCC. Tumor volume and tumor weight of xenografted HCC decreased in nude mice with in vivo knockdown of CDCA2. CONCLUSIONS: CDCA2 triggers proliferative potential in HCC by targeting CCND1 via activating the PI3K/Akt signaling.

Trps1 targets Ccnd1 to regulate mouse Leydig cell proliferation.

BACKGROUND: The tricho-rhino-phalangeal syndrome-1 gene (Trps1) is an atypical GATA family member. Although current studies of Trps1 mainly focus on tumors, whether Trps1 plays a role in the male reproductive system remains unknown. OBJECTIVES: The purpose of this study was to elucidate the function of Trps1 in Leydig cells, indicating its regulatory mechanism on the cell cycle. METHODS: Gene-silencing technology, RNA-seq, RT-qPCR, and western blotting were used to evaluate the function of Trps1 in mouse primary Leydig cells and MLTC-1 cells. In addition, ChIP-base sets and ChIP-qPCR were employed to further assess the regulatory mechanism of Trps1 in MLTC-1 cells. RESULTS: Knockdown of Trps1 in Leydig cells significantly suppressed phosphorylation of Src and Akt and expression of Ccnd1, which was accompanied by impairment of cell proliferative ability. Trps1 may affect the cell cycle through the Src/Akt/Ccnd1 signaling pathway. In addition, Trps1 may bind to the promoter of Srcin1 to regulate its transcription, thus influencing Src phosphorylation levels and the proliferation of Leydig cells. DISCUSSION AND CONCLUSION: Src increases in Leydig cells during pubertal development, suggesting its functional involvement in differentiated adult Leydig cells. Inhibition of the Src/Akt pathway would reduce Ccnd1 expression. In the present study, we found that Trps1 may regulate the phosphorylation level of Src and Akt through Srcin1, targeting Ccnd1 to influence mouse Leydig cell proliferation. These findings shed light on the regulation of Trps1 on cell proliferation and differentiation of mouse Leydig cells.

Fucoxanthin Prevents Colorectal Cancer Development in Dextran Sodium Sulfate-treated Apc Min/+ Mice.

BACKGROUND/AIM: A xanthophyll of fucoxanthin (Fx) is a potential chemopreventive agent. Familial adenomatous polyposis (FAP) is an inherited disease that is associated with a high risk of developing colorectal cancer. However, it remains unclear whether Fx can modify colorectal tumorigenesis in ApcMin/+ mice, a model mouse for human FAP. MATERIALS AND METHODS: We investigated the chemopreventive effect of Fx in dextran sodium sulfate (DSS)-treated ApcMin/+ mice. RESULTS: Administration of Fx in the diet for 5 weeks significantly suppressed the number of colorectal adenocarcinomas in DSS-treated male ApcMin/+ mice, although the treatment did not affect the occurrence of colorectal dysplastic crypts and adenoma in the mice. In addition, Fx down-regulated cyclin D1 expression (0.6-fold) in colorectal mucosa of ApcMin/+ mice when compared with that of the control mice. CONCLUSION: Fx possesses chemopreventive potential against progression of colorectal carcinogenesis in ApcMin/+ mice that receive inflammatory stimuli.

The crosstalk between platelets and body fat: A reverse translational study.

BACKGROUND & AIMS: Our previous study found that platelet counts were positively associated with body fat percentage in human. In the present study, we conducted a reverse translational study to explore the role of platelets in modulating pre-adipocyte proliferation in mice. METHODS: Mouse pre-adipocyte cell line (3T3-L1) and human pre-adipocytes harvested from female subcutaneous fat were used. Pre-adipocytes were co-cultured with platelets or platelet releasate, which were isolated from mice or humans. The cell viability and proliferative ability of the pre-adipocytes were examined by MTT and flow cytometry assays. Western blotting analysis was used to determine the phosphorylation levels of proteins in the mTOR pathway. RESULTS: The number of platelets in the adipose tissues from obese mice was significantly higher than that from lean mice. Platelets and collagen-activated platelet releasate stimulated the proliferation of human pre-adipocytes and 3T3-L1 cells in vitro. Besides, platelets from obese mice were more potent in stimulating pre-adipocyte proliferation than those from lean control mice. Mechanistically, platelets enhanced pre-adipocyte proliferation through the acceleration of cell cycle progression from G0/G1 to S phase cell cycle progression. At the molecular level, platelets promoted pre-adipocyte proliferation through mTOR pathway-mediated upregulation of cyclin D1 expression. CONCLUSION: In conclusion, platelets and platelet releasate play an important role in the proliferation of pre-adipocytes. Our study may provide new clues and the molecular mechanism of the causal pathways between platelets and body fat to explain the finding we observed in population study.

Exercise Partially Rejuvenates Muscle Stem Cells.

Exercise has long been known to extend health and lifespan in humans and other mammals. However, typically exercise is thought to slow the loss of function that accompanies aging. Brett et al. have now shown that exercise restores functional competency to regenerate muscle stem cells (MuSCs) in mice as well as restore a significant portion of the transcriptional signature associated with young MuSCs. The mechanism involves the likely induction of plasma-borne factors that upregulate cell cycle regulator cyclin D1, which otherwise decreases with increasing age. Cyclin D1, in turn, through its noncanonical attenuation of TGF-beta/Smad3 signaling, helps maintain the regenerative capacity of MuSCs, which is lost as TGF-beta signaling increases with age. Interestingly, elevated levels of some proinflammatory regulators including NF-κB, TNF-alpha, and interleukin 6 (IL-6) are also reduced by exercise or ectopic expression of cyclin D1. Importantly, the rejuvenation is not complete, as Notch signaling, which also decreases with age, remains at old levels and the rejuvenative effect is not permanent: wearing off in ∼2 weeks after cessation of exercise. Understanding the limitations of the rejuvenative effect of exercise on MuSCs at the molecular level, including changes in the epigenome such as altered DNA methylation age, will be critical in developing more significant rejuvenative therapies including some for aged people wherein morbidities limit exercise.

CYCLIN-B1/2 and -D1 act in opposition to coordinate cortical progenitor self-renewal and lineage commitment.

The sequential generation of layer-specific cortical neurons requires radial glia cells (RGCs) to precisely balance self-renewal and lineage commitment. While specific cell-cycle phases have been associated with these decisions, the mechanisms linking the cell-cycle machinery to cell-fate commitment remain obscure. Using single-cell RNA-sequencing, we find that the strongest transcriptional signature defining multipotent RGCs is that of G2/M-phase, and particularly CYCLIN-B1/2, while lineage-committed progenitors are enriched in G1/S-phase genes, including CYCLIN-D1. These data also reveal cell-surface markers that allow us to isolate RGCs and lineage-committed progenitors, and functionally confirm the relationship between cell-cycle phase enrichment and cell fate competence. Finally, we use cortical electroporation to demonstrate that CYCLIN-B1/2 cooperate with CDK1 to maintain uncommitted RGCs by activating the NOTCH pathway, and that CYCLIN-D1 promotes differentiation. Thus, this work establishes that cell-cycle phase-specific regulators act in opposition to coordinate the self-renewal and lineage commitment of RGCs via core stem cell regulatory pathways.

CCND1 silencing suppresses liver cancer stem cell differentiation and overcomes 5-Fluorouracil resistance in hepatocellular carcinoma.

OBJECTIVE: Chemoresistance is one of the major barriers in chemotherapy-based hepatocellular carcinoma (HCC) intervention. 5-Fluorouracil (5-Fu) is a widely used as an anticancer drug. Liver cancer stem cells (LCSCs) are considered the origin of tumor recurrence and resistance. CCND1 (Cyclin D1) plays an important role in tumorigenesis and metastasis in multiple cancers including HCC. Herein, this study was designed to explore the role of CCND1 in regulating LCSCs differentiation and 5-Fu resistance in HCC cells. METHODS: The CCND1 mRNA level was examined by qRT-PCR. The protein levels of γ-H2AX (a DNA damage marker) and RAD51 (a DNA repair protein) were examined by Western blot. CD133 was used as a LCSC marker and CD133+ cell percentage in HCC cells was detected by flow cytometry. RESULTS: CCND1 silencing decreased CD133+ cell percentage in HepG2 and SMMC-7721 cells. Furthermore, CCND1 silencing significantly increased protein level of γ-H2AX and decreased that of RAD51 under 5-Fu exposure. Moreover, CCND1 silencing enhanced the sensitivity of HepG2 and SMMC-7721 cells to 5-Fu, which was effectively abrogated by RAD51 upregulation. CONCLUSION: Collectively, CCND1 silencing suppresses LCSCs differentiation and overcomes 5-Fu resistance in HCC.

Cyclin D1 is a mediator of gastrointestinal stromal tumor KIT-independence.

Oncogenic KIT or PDGFRA tyrosine kinase mutations are compelling therapeutic targets in most gastrointestinal stromal tumors (GISTs), and the KIT inhibitor, imatinib, is therefore standard of care for patients with metastatic GIST. However, some GISTs lose expression of KIT oncoproteins, and therefore become KIT-independent and are consequently resistant to KIT-inhibitor drugs. We identified distinctive biologic features in KIT-independent, imatinib-resistant GISTs as a step towards identifying drug targets in these poorly understood tumors. We developed isogenic GIST lines in which the parental forms were KIT oncoprotein-dependent, whereas sublines had loss of KIT oncoprotein expression, accompanied by markedly downregulated expression of the GIST biomarker, protein kinase C-theta (PRKCQ). Biologic mechanisms unique to KIT-independent GISTs were identified by transcriptome sequencing, qRT-PCR, immunoblotting, protein interaction studies, knockdown and expression assays, and dual-luciferase assays. Transcriptome sequencing showed that cyclin D1 expression was extremely low in two of three parental KIT-dependent GIST lines, whereas cyclin D1 expression was high in each of the KIT-independent GIST sublines. Cyclin D1 inhibition in KIT-independent GISTs had anti-proliferative and pro-apoptotic effects, associated with Rb activation and p27 upregulation. PRKCQ, but not KIT, was a negative regulator of cyclin D1 expression, whereas JUN and Hippo pathway effectors YAP and TAZ were positive regulators of cyclin D1 expression. PRKCQ, JUN, and the Hippo pathway coordinately regulate GIST cyclin D1 expression. These findings highlight the roles of PRKCQ, JUN, Hippo, and cyclin D1 as oncogenic mediators in GISTs that have converted, during TKI-therapy, to a KIT-independent state. Inhibitors of these pathways could be effective therapeutically for these now untreatable tumors.

An increase in neural stem cells and olfactory bulb adult neurogenesis improves discrimination of highly similar odorants.

Adult neurogenesis is involved in cognitive performance but studies that manipulated this process to improve brain function are scarce. Here, we characterized a genetic mouse model in which neural stem cells (NSC) of the subventricular zone (SVZ) were temporarily expanded by conditional expression of the cell cycle regulators Cdk4/cyclinD1, thus increasing neurogenesis. We found that supernumerary neurons matured and integrated in the olfactory bulb similarly to physiologically generated newborn neurons displaying a correct expression of molecular markers, morphology and electrophysiological activity. Olfactory performance upon increased neurogenesis was unchanged when mice were tested on relatively easy tasks using distinct odor stimuli. In contrast, intriguingly, increasing neurogenesis improved the discrimination ability of mice when challenged with a difficult task using mixtures of highly similar odorants. Together, our study provides a mammalian model to control the expansion of somatic stem cells that can in principle be applied to any tissue for basic research and models of therapy. By applying this to NSC of the SVZ, we highlighted the importance of adult neurogenesis to specifically improve performance in a challenging olfactory task.