Regulation of cell differentiation by Eph receptor and ephrin signaling

There is increasing evidence that in addition to having major roles in morphogenesis, in some tissues Eph receptor and ephrin signaling regulates the differentiation of cells. In one mode of deployment, cell contact dependent Eph-ephrin activation induces a distinct fate of cells at the interface of their expression domains, for example in early ascidian embryos and in the vertebrate hindbrain. In another mode, overlapping Eph receptor and ephrin expression underlies activation within a cell population, which promotes or inhibits cell differentiation in bone remodelling, neural progenitors and keratinocytes. Eph-ephrin activation also contributes to formation of the appropriate number of progenitor cells by increasing or decreasing cell proliferation. These multiple roles of Eph receptor and ephrin signaling may enable a coupling between morphogenesis and the differentiation and proliferation of cells.     

Regulation of cell differentiation by Eph receptor and ephrin signaling

There is increasing evidence that in addition to having major roles in morphogenesis, in some tissues Eph receptor and ephrin signaling regulates the differentiation of cells. In one mode of deployment, cell contact dependent Eph-ephrin activation induces a distinct fate of cells at the interface of their expression domains, for example in early ascidian embryos and in the vertebrate hindbrain. In another mode, overlapping Eph receptor and ephrin expression underlies activation within a cell population, which promotes or inhibits cell differentiation in bone remodelling, neural progenitors and keratinocytes. Eph-ephrin activation also contributes to formation of the appropriate number of progenitor cells by increasing or decreasing cell proliferation. These multiple roles of Eph receptor and ephrin signaling may enable a coupling between morphogenesis and the differentiation and proliferation of cells.     

Glutamate accelerates RPE cell proliferation through ERK1/2 activation via distinct receptor-specific mechanisms

The proliferation and migration of Retinal Pigment Epithelium cells resulting from an epithelial-mesenchymal transition plays a key role in proliferative vitreoretinopathy, which leads to retinal detachment and the loss of vision. In neurons, glutamate has been shown to activate the Ras/Raf/MEK/ERK cascade, which participates in the regulation of proliferation, differentiation, and survival processes. Although glutamate-stimulation and the activation of ERK1/2 by different stimuli have been shown to promote RPE cell proliferation, the signaling pathway(s) linking these effects has not been established. We analyzed the molecular mechanisms leading to glutamate-induced proliferation by determining ERK1/2 and CREB phoshporylation in chick RPE cells in primary culture and the human-derived RPE cell line ARPE-19. This study shows for the first time, that glutamate promotes RPE cell proliferation by activating two distinct signaling pathways linked to selective glutamate receptor subtypes. Results demonstrate that glutamate stimulates RPE cell proliferation as well as ERK and CREB phosphorylation. These effects were mimicked by the mGluR agonist ACPD and by NMDA, and were prevented by the respective receptor inhibitors MCPG and MK-801, indicating a cause-effect relationship between these processes. Whereas mGluR promoted proliferation by activating the MEK/ERK/CREB cascade, NMDA stimulated proliferation through the MEK-independent activation of Ca(2+)/calmodulin-dependent kinases. The blockage of both signaling pathways to proliferation by KN-62 suggests the involvement of CaMKs in the control of glutamate-induced proliferation at a common step, downstream of CREB, possibly the regulation of cell cycle progression. Based on these findings, the participation of glutamate in the development of PVR can be considered.     

Targeted Ablation of Crb1 and Crb2 in Retinal Progenitor Cells Mimics Leber Congenital Amaurosis

Development in the central nervous system is highly dependent on the regulation of the switch from progenitor cell proliferation to differentiation, but the molecular and cellular events controlling this process remain poorly understood. Here, we report that ablation of Crb1 and Crb2 genes results in severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of CRB1 function. We show that the levels of CRB1 and CRB2 proteins are crucial for mouse retinal development, as they restrain the proliferation of retinal progenitor cells. The lack of these apical proteins results in altered cell cycle progression and increased number of mitotic cells leading to an increased number of late-born cell types such as rod photoreceptors, bipolar and Müller glia cells in postmitotic retinas. Loss of CRB1 and CRB2 in the retina results in dysregulation of target genes for the Notch1 and YAP/Hippo signaling pathways and increased levels of P120-catenin. Loss of CRB1 and CRB2 result in altered progenitor cell cycle distribution with a decrease in number of late progenitors in G1 and an increase in S and G2/M phase. These findings suggest that CRB1 and CRB2 suppress late progenitor pool expansion by regulating multiple proliferative signaling pathways.     

A requirement for fibroblast growth factor in regulation of skeletal muscle growth and differentiation cannot be replaced by activation of platelet-derived growth factor signaling pathways.

The distinct effects of cytokines on cellular growth and differentiation suggest that specific signaling pathways mediate these diverse biological activities. Fibroblast growth factors (FGFs) are well-established inhibitors of skeletal muscle differentiation and may operate via activation of specific signaling pathways distinct from recently identified mitogen signaling pathways. We examined whether platelet-derived growth factor (PDGF)-activated signaling pathways are sufficient to mediate FGF-dependent repression of myogenesis by introducing the PDGF beta receptor into a mouse skeletal muscle cell line. Addition of PDGF-BB to cells expressing the PDGF beta receptor activated the PDGF beta receptor tyrosine kinase, stimulated mitogen-activated protein (MAP) kinase, and increased the steady-state levels of junB and c-fos mRNAs. Despite the activation of these intracellular signaling molecules, PDGF beta receptor activation elicited no detectable effect on cell proliferation or differentiation. In contrast to PDGF-BB, addition of FGF-2 to myoblasts activated signaling pathways that resulted in DNA synthesis and repression of differentiation. Because of the low number of endogenous FGF receptors expressed, FGF-stimulated signaling events, including tyrosine phosphorylation and activation of MAP kinase, could be detected only in cells expressing higher levels of a transfected FGF receptor cDNA. As the PDGF beta receptor- and FGF receptor-stimulated signaling pathways yield different biological responses in these skeletal muscle cells, we hypothesize that FGF-mediated repression of skeletal muscle differentiation activates signaling pathways distinct from those activated by the PDGF beta receptor. Activation of PDGF beta receptor tyrosine kinase activity, stimulation of MAP kinase, and upregulation of immediate-early gene expression are not sufficient to repress skeletal muscle differentiation.     

Retinoic acid induces CDK inhibitors and growth arrest specific (Gas) genes in neural crest cells

Retinoic acid (RA), the active metabolite of vitamin A, regulates cellular growth and differentiation during embryonic development. In excess, this vitamin is also highly teratogenic to animals and humans. The neural crest is particularly sensitive to RA, and high levels adversely affect migration, proliferation and cell death. We investigated potential gene targets of RA associated with neural crest proliferation by determining RA-mediated changes in gene expression over time, using microarrays. Statistical analysis of the top ranked RA-regulated genes identified modest changes in multiple genes previously associated with cell cycle control and proliferation including the cyclin-dependent kinase inhibitors Cdkn1a (p21), Cdkn2b (p15(INK4b)), and Gas3/PMP22. The expression of p21 and p15(INK4b) contribute to decreased proliferation by blocking cell cycle progression at G1-S. This checkpoint is pivotal to decisions regulating proliferation, apoptosis, or differentiation. We have also confirmed the overexpression of Gas3/PMP22 in RA-treated neural crests, which is associated with cytoskeletal changes and increased apoptosis. Our results suggest that increases in multiple components of diverse regulatory pathways have an overall cumulative effect on cellular decisions. This heterogeneity contributes to the pleiotropic effects of RA, specifically those affecting proliferation and cell death.     

RSPO1/β-catenin signaling pathway regulates oogonia differentiation and entry into meiosis in the mouse fetal ovary

Differentiation of germ cells into male gonocytes or female oocytes is a central event in sexual reproduction. Proliferation and differentiation of fetal germ cells depend on the sex of the embryo. In male mouse embryos, germ cell proliferation is regulated by the RNA helicase Mouse Vasa homolog gene and factors synthesized by the somatic Sertoli cells promote gonocyte differentiation. In the female, ovarian differentiation requires activation of the WNT/β-catenin signaling pathway in the somatic cells by the secreted protein RSPO1. Using mouse models, we now show that Rspo1 also activates the WNT/β-catenin signaling pathway in germ cells. In XX Rspo1(-/-) gonads, germ cell proliferation, expression of the early meiotic marker Stra8, and entry into meiosis are all impaired. In these gonads, impaired entry into meiosis and germ cell sex reversal occur prior to detectable Sertoli cell differentiation, suggesting that β-catenin signaling acts within the germ cells to promote oogonial differentiation and entry into meiosis. Our results demonstrate that RSPO1/β-catenin signaling is involved in meiosis in fetal germ cells and contributes to the cellular decision of germ cells to differentiate into oocyte or sperm.     

Convergence of multiple signaling pathways is required to coordinately up-regulate mtDNA and mitochondrial biogenesis during T cell activation

The quantity and activity of mitochondria vary dramatically in tissues and are modulated in response to changing cellular energy demands and environmental factors. The amount of mitochondrial DNA (mtDNA), which encodes essential subunits of the oxidative phosphorylation complexes required for cellular ATP production, is also tightly regulated, but by largely unknown mechanisms. Using murine T cells as a model system, we have addressed how specific signaling pathways influence mitochondrial biogenesis and mtDNA copy number. T cell receptor (TCR) activation results in a large increase in mitochondrial mass and membrane potential and a corresponding amplification of mtDNA, consistent with a vital role for mitochondrial function for growth and proliferation of these cells. Independent activation of protein kinase C (via PMA) or calcium-related pathways (via ionomycin) had differential and sub-maximal effects on these mitochondrial parameters, as did activation of naïve T cells with proliferative cytokines. Thus, the robust mitochondrial biogenesis response observed upon TCR activation requires synergy of multiple downstream signaling pathways. One such pathway involves AMP-activated protein kinase (AMPK), which we show has an unprecedented role in negatively regulating mitochondrial biogenesis that is mammalian target of rapamycin (mTOR)-dependent. That is, inhibition of AMPK after TCR signaling commences results in excessive, but uncoordinated mitochondrial proliferation. Thus mitochondrial biogenesis is not under control of a single master regulatory circuit, but rather requires the convergence of multiple signaling pathways with distinct downstream consequences on the organelle’s structure, composition, and function.     

小鼠正常黑素细胞和小鼠B16黑素瘤细胞的环状RNAs表达谱

黑素瘤是一种多发于皮肤的恶性肿瘤,因其侵袭性强,预后差等特点一直是科研人员关注的热点。环状RNAs(circRNAs)是一种新型内源性非编码RNA,广泛参与动物生长发育、细胞分化和信号转导等生理过程,但circRNAs在黑素瘤细胞内的分子机制尚未被充分解析。本研究以小鼠(C57BL/6J)正常黑素细胞及B16黑素瘤细胞为研究对象,采用二代测序技术分析两种细胞间circRNAs表达特性。测序结果显示,小鼠正常黑素细胞和黑素瘤细胞中共有851个circRNAs,其中195个差异表达circRNAs(DECs)。GO及KEGG数据库注释发现,DECs的来源基因主要参与细胞周期(cell cycle)、紧密结合(tight junction)、Rap1信号通路(Rap1 signaling pathway)、TGF-beta信号通路(TGF-beta signaling pathway)等与细胞增殖、迁移相关的信号通路;探究发现,黑素瘤细胞中显著性高表达的circE2F5(circ-3:14578602|14606309)通过上调E2F5的表达促进黑素瘤细胞增殖。circRNA靶基因预测发现,...     

Src Family Kinases Are Required for Prolactin Induction of Cell Proliferation

Prolactin (PRL) is a pleiotropic cytokine promoting cellular proliferation and differentiation. Because PRL activates the Src family of tyrosine kinases (SFK), we have studied the role of these kinases in PRL cell proliferation signaling. PRL induced [(3)H]thymidine incorporation upon transient transfection of BaF-3 cells with the PRL receptor. This effect was inhibited by cotransfection with the dominant negative mutant of c-Src (K>A295/Y>F527, SrcDM). The role of SFK in PRL-induced proliferation was confirmed in the BaF-3 PRL receptor-stable transfectant, W53 cells, where PRL induced Fyn and Lyn activation. The SFK-selective inhibitors PP1/PP2 and herbimycin A blocked PRL-dependent cell proliferation by arresting the W53 cells in G1, with no evident apoptosis. In parallel, PP1/PP2 inhibited PRL induction of cell growth-related genes c-fos, c-jun, c-myc, and odc. These inhibitors have no effect on PRL-mediated activation of Ras/Mapk and Jak/Start pathways. In contrast, they inhibited the PRL-dependent stimulation of the SFKs substrate Sam68, the phosphorylation of the tyrosine phosphatase Shp2, and the PI3K-dependent Akt and p70S6k serine kinases. Consistently, transient expression of SrcDM in W53 cells also blocked PRL activation of Akt. These results demonstrate that activation of SFKs is required for cell proliferation induced by PRL.     

NFκB signaling regulates embryonic and adult neurogenesis

Both embryonic and adult neurogenesis involves the self-renewal/proliferation,survival,migration and lineage differentiation of neural stem/progenitor cells.Such dynamic process is tightly regulated by...     

The role of Wnt/β‐catenin signaling in proliferation and regeneration of the developing basilar papilla and lateral line

Canonical Wnt/β‐catenin signaling has been implicated in multiple developmental events including the regulation of proliferation, cell fate, and differentiation. In the inner ear, Wnt/β‐catenin signaling is required from the earliest stages of otic placode specification through the formation of the mature cochlea. Within the avian inner ear, the basilar papilla (BP), many Wnt pathway components are expressed throughout development. Here, using reporter constructs for Wnt/β‐catenin signaling, we show that this pathway is active throughout the BP (E6‐E14) in both hair cells (HCs) and supporting cells. To characterize the role of Wnt/β‐catenin activity in developing HCs, we performed gain‐ and loss‐of‐function experiments in vitro and in vivo in the chick BP and zebrafish lateral line systems, respectively. Pharmacological inhibition of Wnt signaling in the BP and lateral line neuromasts during the periods of proliferation and HC differentiation resulted in reduced proliferation and decreased HC formation. Conversely, pharmacological activation of this pathway significantly increased the number of HCs in the lateral line and BP. Results demonstrated that this increase was the result of up‐regulated cell proliferation within the Sox2‐positive cells of the prosensory domains. Furthermore, Wnt/β‐catenin activation resulted in enhanced HC regeneration in the zebrafish lateral line following aminoglycoside‐induced HC loss. Combined, our data suggest that Wnt/β‐catenin signaling specifies the number of cells within the prosensory domain and subsequently the number of HCs. This ability to induce proliferation suggests that the modulation of Wnt/β‐catenin signaling could play an important role in therapeutic HC regeneration. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 438–456, 2014     

Epac1‐induced cellular proliferation in prostate cancer cells is mediated by B‐Raf/ERK and mTOR signaling cascades

cAMP‐dependent, PKA‐independent effects on cell proliferation are mediated by cAMP binding to EPAC and activation of Rap signaling. In this report, we employed the analogue 8‐CPT‐2‐O‐Me‐cAMP to study binding to EPAC and subsequent activation of B‐Raf/ERK and mTOR signaling in human cancer cells. This compound significantly stimulated DNA synthesis, protein synthesis, and cellular proliferation of human 1‐LN prostate cancer cells. By study of phosphorylation‐dependent activation, we demonstrate that EPAC‐mediated cellular effects require activation of the B‐Raf/ERK and mTOR signaling cascades. RNAi directed against EPAC gene expression as well as inhibitors of ERK, PI 3‐kinase, and mTOR were employed to further demonstrate the role of these pathways in regulating prostate cancer cell proliferation. These studies were then extended to several other human prostate cancer cell lines and melanoma cells with comparable results. We conclude that B‐Raf/ERK and mTOR signaling play an essential role in cAMP‐dependent, but PKA‐independent, proliferation of cancer cells. J. Cell. Biochem. 108: 998–1011, 2009. © 2009 Wiley‐Liss, Inc.