Adult neurogenesis (i.e., proliferation and differentiation of neuronal precursors in the adult brain) is responsible for adding new neurons in the dentate gyrus of the hippocampus and in the olfactory bulb. We describe herein that adult mice mutated in the cell cycle regulatory gene Ccnd2, encoding cyclin D2, lack newly born neurons in both of these brain structures. In contrast, genetic ablation of cyclin D1 does not affect adult neurogenesis. Furthermore, we show that cyclin D2 is the only D-type cyclin (out of D1, D2, and D3) expressed in dividing cells derived from neuronal precursors present in the adult hippocampus. In contrast, all three cyclin D mRNAs are present in the cultures derived from 5-day-old hippocampi, when developmental neurogenesis in the dentate gyrus takes place. Thus, our results reveal the existence of molecular mechanisms discriminating adult versus developmental neurogeneses.

Hepatitis B virus (HBV) infection is the leading cause of liver diseases. However, the molecular mechanisms of HBV infection and carcinogenesis have not been fully elucidated. In this study, we found that cyclin D2 was upregualted in HBV-expressing cells and liver tissues of HBV-transgenic mice. Gene silencing of cyclin D2 inhibited HBV DNA replicative intermediates, 3.5 kb mRNA, core protein level, as well as the secretions of HBsAg and HBeAg. On the contrary, overexpression of cyclin D2 promoted HBV replication. Furthermore, cyclin D2 regulated HBV replication by enhancing the activity of HBV core and Sp1 promoters by targeting transcription factor CREB2. Silencing of CREB2 abolished enhancement of HBV replication induced by cyclin D2. Together, our study has uncovered a positive role of cyclin D2 in HBV replication. It is conceivable that therapeutic application of cyclin D2 inhibitor in HBV infection therapy.

Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma with poor long-term overall survival. Currently, MCL research and development of potential cures is hampered by the lack of good in vivo models. MCL is characterized by recurrent translocations of CCND1 or CCND2, resulting in overexpression of the cell cycle regulators cyclin D1 or D2, respectively. Here, we show, for the first time, that hematopoiesis-specific activation of cyclin D2 is sufficient to drive murine MCL-like lymphoma development. Furthermore, we demonstrate that cyclin D2 overexpression can synergize with loss of p53 to form aggressive and transplantable MCL-like lymphomas. Strikingly, cyclin D2-driven lymphomas display transcriptional, immunophenotypic, and functional similarities with B1a B cells. These MCL-like lymphomas have B1a-specific B cell receptors (BCRs), show elevated BCR and NF-κB pathway activation, and display increased MALT1 protease activity. Finally, we provide preclinical evidence that inhibition of MALT1 protease activity, which is essential for the development of early life-derived B1a cells, can be an effective therapeutic strategy to treat MCL.

Diabetes results from an inadequate mass of functional β cells, due to either β cell loss caused by autoimmune destruction (type I diabetes) or β cell failure in response to insulin resistance (type II diabetes). Elucidating the mechanisms that regulate β cell mass may be key to developing new techniques that foster β cell regeneration as a cellular therapy to treat diabetes. While previous studies concluded that cyclin D2 is required for postnatal β cell self-renewal in mice, it is not clear if cyclin D2 is sufficient to drive β cell self-renewal. Using transgenic mice that overexpress cyclin D2 specifically in β cells, we show that cyclin D2 overexpression increases β cell self-renewal post-weaning and results in increased β cell mass. β cells that overexpress cyclin D2 are responsive to glucose stimulation, suggesting they are functionally mature. β cells that overexpress cyclin D2 demonstrate an enhanced regenerative capacity after injury induced by streptozotocin toxicity. To understand if cyclin D2 overexpression is sufficient to drive β cell self-renewal, we generated a novel mouse model where cyclin D2 is only expressed in β cells of cyclin D2-/- mice. Transgenic overexpression of cyclin D2 in cyclin D2-/- β cells was sufficient to restore β cell mass, maintain normoglycaemia, and improve regenerative capacity when compared with cyclin D2-/- littermates. Taken together, our results indicate that cyclin D2 is sufficient to regulate β cell self-renewal and that manipulation of its expression could be used to enhance β cell regeneration.

Sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables, induces potent anti-proliferative effects in prostate cancer cells. One mechanism that may contribute to the anti-proliferative effects of SFN is the modulation of epigenetic marks, such as inhibition of histone deacetylase (HDAC) enzymes. However, the effects of SFN on other common epigenetic marks such as DNA methylation are understudied. Promoter hyper-methylation of cyclin D2, a major regulator of cell cycle, is correlated with prostate cancer progression, and restoration of cyclin D2 expression exerts anti-proliferative effects on LnCap prostate cancer cells. Our study aimed to investigate the effects of SFN on DNA methylation status of cyclin D2 promoter, and how alteration in promoter methylation impacts cyclin D2 gene expression in LnCap cells. We found that SFN significantly decreased the expression of DNA methyltransferases (DNMTs), especially DNMT1 and DNMT3b. Furthermore, SFN significantly decreased methylation in cyclin D2 promoter regions containing c-Myc and multiple Sp1 binding sites. Reduced methlyation of cyclin D2 promoter corresponded to an increase in cyclin D2 transcript levels, suggesting that SFN may de-repress methylation-silenced cyclin D2 by impacting epigenetic pathways. Our results demonstrated the ability of SFN to epigenetically modulate cyclin D2 expression, and provide novel insights into the mechanisms by which SFN may regulate gene expression as a prostate cancer chemopreventive agent.

Recent reports have shown that cannabinoid 1 receptors (CB1Rs) are expressed in pancreatic β cells, where they induce cell death and cell cycle arrest by directly inhibiting insulin receptor activation. Here, we report that CB1Rs regulate the expression of the anti-apoptotic protein Bcl-2 and cell cycle regulator cyclin D2 in pancreatic β cells. Treatment of MIN6 and βTC6 cells with a synthetic CB1R agonist, WIN55,212-2, led to a decrease in the expression of Bcl-2 and cyclin D2, in turn inducing cell cycle arrest in G0/G1 phase and caspase-3-dependent apoptosis. Additionally, genetic deletion and pharmacological blockade of CB1Rs after injury in mice led to increased levels of Bcl-2 and cyclin D2 in pancreatic β cells. These findings provide evidence for the involvement of Bcl-2 and cyclin D2 mediated by CB1Rs in the regulation of β-cell survival and growth, and will serve as a basis for developing new therapeutic interventions to enhance β-cell function and growth in diabetes.

FOXL2 is a transcription factor that is essential for ovarian development. Somatic mutations of FOXL2 are associated with ovarian granulosa cell tumorigenesis. In the present study, the expression of FOXL2 was suppressed by microRNAs using the Ago2 knockdown method in COV434 cells. Online bioinformatics tools were utilized to predict that FOXL2 expression may be repressed by miR-30 family members, and dual luciferase assay and western blotting were performed to demonstrate that FOXL2 is a target gene of miR-30a, which is relatively abundant in COV434 cells. Furthermore, miR-30a overexpression upregulates BCL2A1, IER3 and cyclin D2 expression by inhibiting FOXL2. miR-30a is known to function as a tumor suppressor in breast cancer, small cell lung cancer and colorectal carcinoma; however, the present study revealed an opposing function of miR-30a as an oncogene.

We have recently cloned a novel splice variant of cyclin D2 termed as cycD2SV. CycD2SV overexpression in several immortalized cell lines led to formation of ubiquitinated protein aggregates accompanied by a significant decrease in cell proliferation. Based on immuno co-localization and ultrastructural analysis experiments, cycD2SV protein aggregates were frequently found in various subcellular compartments such as endosomes, autophagosomes, lysosomes and the microtubule organizing centre. Secondary structure analysis revealed that the amino terminal α-helix in cycD2SV is not tightly packed with the cyclin box suggesting a misfolded conformation compared to other cyclins. Deletion analysis suggests that 1-53 amino acid region of cycD2SV may be required for protein aggregation and 54-136 amino acid region may mediate cell cycle inhibition. Based on co-immunoprecipitation experiments, we have shown that cycD2SV binds to cycD2 as well as CDK4. In addition, gene expression analysis demonstrated an upregulation in GADD45α and dynamin 2 mRNA levels in cycD2SV overexpressing cells. These two proteins are known to play critical roles in the DNA damage response and apoptosis pathways. TUNEL experiments were negative for apoptosis, however, cycD2SV expressing cells were more sensitive to cell death induced by external stressors such as trypsinization. Collectively our results suggest that cycD2SV mediates cell cycle inhibition by sequestering endogenous cell cycle proteins, such as cycD2 and CDK4, and possibly targeting them for ubiquitin mediated protein degradation.

MiR-198 has been considered as an inhibitor of cell proliferation, invasion, migration and a promoter of apoptosis in most cancer cells, while its effect on non-cancer cells is poorly understood.

D-cyclin proteins play a central role in cell-cycle regulation and are involved in the pathogenesis of lymphomas. In mantle-cell lymphoma, the t(11;14) translocation leads to overexpression of cyclin-D1, in addition to which cyclin-D1-negative mantle-cell lymphoma that overexpress cyclin-D2 or D3 have also been described. Although cyclin-D2 and D3 have been implicated in the prognosis of specific lymphoma subtypes, a thorough characterization of D-cyclin protein expression in human hematolymphoid neoplasia has not been reported. To evaluate the tissue expression patterns of D-cyclins, particularly D2 and D3, in normal and neoplastic hematolymphoid tissues, we optimized the commercially available antibodies for D-cyclins for use on paraffin-embedded tissue and stained tissue microarrays of over 700 patient samples. Our results show that cyclin-D2 and D3 proteins are expressed in many more lymphoma subtypes than cyclin-D1. Cyclin-D1, D2 and D3 were expressed in 100, 22 and 6% of mantle-cell lymphomas and 2, 49 and 20% of diffuse large B-cell lymphomas. Fluorescence in situ hybridization studies confirmed the presence of the CCND1/IGH translocation in the majority of mantle-cell lymphoma, but not in diffuse large B-cell lymphoma that expressed cyclin-D1 protein. In addition, a subset of follicular, marginal zone, lymphoplasmacytic, lymphoblastic, classical Hodgkin, mature T-cell and natural killer cell lymphomas and acute myeloid leukemias also expressed cyclin-D2 and D3. These data support the hypothesis that dysregulation of cell-cycle control by D-cyclins contribute to the pathogenesis of hematolymphoid neoplasia, and suggest a potential role for these proteins in the prognostic and therapeutic aspects of these diseases. For diagnostic purposes, however, the expression of D-cyclin proteins should be interpreted with caution in the subclassification of lymphoma types.

B-1 lymphocytes represent a distinct B cell subset with characteristic features that include self-renewing capacity and unusual mitogenic responses. B-1 cells differ from conventional B cells in terms of the consequences of phorbol ester treatment: B-1 cells rapidly enter S phase in response to phorbol ester alone, whereas B-2 cells require a calcium ionophore in addition to phorbol ester to trigger cell cycle progression. To address the mechanism underlying the varied proliferative responses of B-1 and B-2 cells, we evaluated the expression and activity of the G1 cell cycle regulator, cyclin D2, and its associated cyclin-dependent kinases (Cdks). Cyclin D2 expression was upregulated rapidly, within 2-4 h, in phorbol ester-stimulated B-1 cells, in a manner dependent on intact transcription/translation, but was not increased in phorbol ester- stimulated B-2 cells. Phorbol ester-stimulated cyclin D2 expression was accompanied by the formation of cyclin D2-Cdk4, and, to a lesser extent, cyclin D2-Cdk6, complexes; cyclin D2- containing complexes were found to be catalytically functional, in terms of their ability to phosphorylate exogenous Rb in vitro and to specifically phosphorylate endogenous Rb on serine780 in vivo. These results strongly suggest that the rapid induction of cyclin D2 by a normally nonmitogenic phorbol ester stimulus is responsible for B-1 cell progression through G1 phase. The ease and rapidity with which cyclin D2 responds in B-1 cells may contribute to the proliferative features of this subset.

In the current study, we analyzed the effects of the systemic inflammatory response (SIR) and amyloid β (Aβ) peptide on the expression of genes encoding cyclins and cyclin-dependent kinase (Cdk) in: (i) PC12 cells overexpressing human beta amyloid precursor protein (βAPP), wild-type (APPwt-PC12), or carrying the Swedish mutantion (APPsw-PC12); (ii) the murine hippocampus during SIR; and (iii) Alzheimer's disease (AD) brain. In APPwt-PC12 expression of cyclin D2 (cD2) was exclusively reduced, and in APPsw-PC12 cyclins cD2 and also cA1 were down-regulated, but cA2, cB1, cB2, and cE1 were up-regulated. In the SIR cD2, cB2, cE1 were found to be significantly down-regulated and cD3, Cdk5, and Cdk7 were significantly up-regulated. Cyclin cD2 was also found to be down-regulated in AD neocortex and hippocampus. Our novel data indicate that Aβ peptide and inflammation both significantly decreased the expression of cD2, suggesting that Aβ peptides may also contribute to downregulation of cD2 in AD brain.

Cyclin D2 is involved in the pathology of vascular complications of type 2 diabetes mellitus (T2DM). This study investigated the role of cyclin-D2-regulated miRNAs in endothelial cell proliferation of T2DM. Results showed that higher glucose concentration (4.5 g/l) significantly promoted the proliferation of rat aortic endothelial cells (RAOECs), and significantly increased the expression of cyclin D2 and phosphorylation of retinoblastoma 1 (p-RB1) in RAOECs compared with those under low glucose concentration. The cyclin D2-3' untranslated region is targeted by miR-98, as demonstrated by miRNA analysis software. Western blot also confirmed that cyclin D2 and p-RB1 expression was regulated by miR-98. The results indicated that miR-98 treatment can induce RAOEC apoptosis. The suppression of RAOEC growth by miR-98 might be related to regulation of Bcl-2, Bax and Caspase 9 expression. Furthermore, the expression levels of miR-98 decreased in 4.5 g/l glucose-treated cells compared with those treated by low glucose concentration. Similarly, the expression of miR-98 significantly decreased in aortas of established streptozotocin (STZ)-induced diabetic rat model compared with that in control rats; but cyclin D2 and p-RB1 levels remarkably increased in aortas of STZ-induced diabetic rats compared with those in healthy control rats. In conclusion, this study demonstrated that high glucose concentration induces cyclin D2 up-regulation and miR-98 down-regulation in the RAOECs. By regulating cyclin D2, miR-98 can inhibit human endothelial cell growth, thereby providing novel therapeutic targets for vascular complication of T2DM.

MicroRNA-1 (miRNA-1) has been long viewed as a muscle-specific miRNA and plays a critical role in myocardium and cardiomyocytes by controlling myocyte growth and rhythm. We identified that miRNA-1 is expressed in cardiac fibroblasts, which are one of the major non-muscle cell types in myocardium and are responsible for cardiac fibrosis in pathological conditions. In this study, we aimed to investigate the effect and mechanism of action of miRNA-1 on cardiac fibroblast proliferation. Subcutaneous angiotensin II (Ang II) infusion via osmotic minipumps for 4 weeks was used to induce myocardial interstitial fibrosis in male Sprague-Dawley rats. MiRNA-1 expression was significantly down-regulated by 68% in freshly isolated ventricular fibroblasts from Ang II-infused rats than that from control rats. Similar results were obtained in adult rat ventricular fibroblasts that were stimulated in culture by Ang II or TGFβ for 48 h. Functionally, overexpression of miRNA-1 inhibited fibroblast proliferation, whereas knockdown of endogenous miRNA-1 increased fibroblast proliferation. We then identified and validated cyclin D2 and cyclin-dependent kinase 6 (CDK6) as direct targets of miRNA-1 in cardiac fibroblasts using biochemical assays. Moreover, we showed that the inhibitory effects of miRNA-1 on cardiac fibroblast proliferation can be blunted by overexpression of its target, cyclin D2. In conclusion, our findings demonstrate miRNA-1 expression and regulation in adult ventricular fibroblasts, where it acts as a novel negative regulator of adult cardiac fibroblast proliferation that is at least partially mediated by direct targeting of two cell cycle regulators. Our results expand the understanding of the regulatory roles of miRNA-1 in cardiac cells (i.e., from myocytes to a major non-muscle cells in the heart).

Previous studies have reported that miR-615 exerts a tumor suppressor role in some tumors, such as esophageal squamous cell carcinoma and non-small cell lung cancer. However, the role of miR-615 in prostate cancer has not been defined. Here we found that miR-615 was downregulated in prostate cancer tissues and cell lines. Overexpression of miR-615 in PC-3 cells significantly inhibited cellular proliferation, migration, and invasion. Moreover, overexpression of miR-615 delayed tumor growth in vivo. In terms of mechanism, we found that cyclin D2 (CCND2) is a target gene of miR-615 in prostate cancer. We showed that miR-615 could bind to the 3'-UTR region of CCND2 mRNA and inhibit its expression. There was a negative correlation between the expression of miR-615 and CCND2 in prostate cancer tissues. Moreover, restoration of cyclin D2 abolished the inhibitory effects of miR-615 on the proliferation, migration, and invasion of prostate cancer cells. Taken together, our study identified miR-615 as a tumor suppressor by targeting cyclin D2 in prostate cancer.

CD274 (programmed death ligand 1, also known as B7H1) is expressed in both solid tumors and hematologic malignancies and is of critical importance for the escape of tumor cells from immune surveillance by inhibiting T cell function via its receptor, programmed death 1 (PD-1). Increasing evidence indicates that functional monoclonal antibodies of CD274 may potently enhance the antitumor effect in many cancers. However, the role of CD274 in leukemia-initiating cells (LICs) remains largely unknown.

MicroRNAs (miRNAs/miRs) serve important roles in the chemotherapeutic effect of anticancer drugs. To investigate the roles of miRNAs in cisplatin‑induced suppression of lung adenocarcinoma cell proliferation, A549 cells were treated with different concentrations of cisplatin. An MTT assay demonstrated that cisplatin inhibited A549 cell proliferation in a dose‑dependent manner. Cisplatin induced cell apoptosis and inhibited cell migration by increasing the levels of miR‑93, miR‑26a and miR‑26b. Furthermore, as an upstream factor, miR‑93 was proposed to regulate cyclin D2 expression in miR‑93‑transfected A549 cells. Cisplatin also induced Bcl‑2‑associated X protein expression, and decreased that of Bcl‑2 and c‑Myc in lung adenocarcinoma cells. In vivo analysis further supported that cisplatin inhibited lung adenocarcinoma cell growth by regulating cyclin D2 and miR‑93 expression. In conclusion, our findings demonstrated that cisplatin could effectively inhibit lung adenocarcinoma cell proliferation by decreasing cyclin D2 expression via miR‑93.

p75NTR is a member of the tumor necrosis superfamily of proteins which is variably associated with induction of apoptosis and proliferation. Cyclin D2 is one of the mediators of cellular progression through G1 phase of the cell cycle. The present study demonstrates the inverse relationship between expression of cyclin D2 and expression of p75NTR in PC12 cells. Induction of p75NTR expression in p75NTR-negative PC12 cells results in downregulation of cyclin D2; suppression of p75NTR expression with siRNA in native PC12 cells results in upregulation of cyclin D2. The effects of p75NTR on cyclin D2 expression are mimicked in p75NTR-negative cells by transfection with the intracellular domain of p75NTR. Cyclin-D2-positive PC12 cell cultures grow more slowly than cyclin-D2-negative cultures, and induction of expression of cyclin D2 slows the culture growth rate of cyclin-D2-negative cells. Finally, subcutaneous murine xenografts of cyclin-D2-negative, p75NTR-positive PC12 cells more frequently and more rapidly produce tumors than the analogous xenografts of cyclin-D2-positive, p75NTR-negative cells. These results suggest that p75NTR suppresses cyclin D2 expression in PC12 cells by a mechanism distinct from its function as a nerve growth factor receptor and that cyclin D2 expression decreases cell culture and xenografted tumor growth.

Elevated activity at the output stage of the anterior hippocampus has been described as a physiological endophenotype of schizophrenia, and its development maps onto the transition from the prodromal to the psychotic state. Interventions that halt the spreading glutamatergic over-activity in this region and thereby the development of overt schizophrenia could be promising therapies. However, animal models with high construct validity to support such pre-clinical development are scarce. The Cyclin-D2 knockout (CD2-KO) mouse model shows a hippocampal parvalbumin-interneuron dysfunction, and its pattern of hippocampal over-activity shares similarities with that seen in prodromal patients. Conducting a comprehensive phenotyping of CD2-KO mice, we found that they displayed novelty-induced hyperlocomotion (a rodent correlate of positive symptoms of schizophrenia), that was largely resistant against D1- and D2-dopamine-receptor antagonism, but responsive to the mGluR2/3-agonist LY379268. In the negative symptom domain, CD2-KO mice showed transiently reduced sucrose-preference (anhedonia), but enhanced interaction with novel mice and objects, as well as normal nest building and incentive motivation. Also, unconditioned anxiety, perseveration, and motor-impulsivity were unaltered. However, in the cognitive domain, CD2-knockouts showed reduced executive function in assays of rule-shift and rule-reversal learning, and also an impairment in working memory, that was resistant against LY379268-treatment. In contrast, sustained attention and forms of spatial and object-related memory that are mediated by short-term habituation of stimulus-specific attention were intact. Our results suggest that CD2-KO mice are a valuable model in translational research targeted at the pharmacoresistant cognitive symptom domain in causal relation to hippocampal over-activity in the prodrome-to-psychosis transition.

Ectopic expression of Myc induces Cdk2 kinase activity in quiescent cells and antagonizes association of p27(kip1) with Cdk2. The target gene(s) by which Myc mediates this effect is largely unknown. We now show that p27 is rapidly and transiently sequestered by cyclin D2-Cdk4 complexes upon activation of Myc and that cyclin D2 is a direct target gene of Myc. The cyclin D2 promoter is repressed by Mad-Max complexes and de-repressed by Myc via a single highly conserved E-box element. Addition of trichostatin A to quiescent cells mimics activation of Myc and induces cyclin D2 expression, suggesting that cyclin D2 is repressed in a histone deacetylase-dependent manner in quiescent cells. Inhibition of cyclin D2 function in established cell lines, either by ectopic expression of p16 or by antibody injection, inhibits Myc-dependent dissociation of p27 from Cdk2 and Myc-induced cell cycle entry. Primary mouse fibroblasts that are cyclin D2-deficient undergo accelerated senescence in culture and are not immortalized by Myc; induction of apoptosis by Myc is unimpaired in such cells. Our data identify a downstream effector pathway that links Myc directly to cell cycle progression.