Transgenic expression of the p16(INK4a) cyclin-dependent kinase inhibitor leads to enhanced apoptosis and differentiation arrest of CD4-CD8- immature thymocytes

In the thymus, T cell development proceeds by successive steps of differentiation, expansion, and selection. Control of thymocyte proliferation is critical to insure the full function of the immune system and to prevent T cells from transformation. Deletion of the cell cycle inhibitor p16(INK4a) is frequently observed in human T cell neoplasias and, in mice, gene targeted inactivation of the Ink4a locus enhances thymocyte expansion and predisposes mutant animal to tumorigenesis. Here, we investigate the mechanism by which p16(Ink4a) controls thymocyte development by analyzing transgenic mice expressing the human p16(INK4a) into the T cell lineage. We show that forced expression of p16(INK4a) in thymocytes blocked T cell differentiation at the early CD4-CD8-CD3-CD25+ stage without significantly affecting the development of gammadelta T cells. Pre-TCR function was mimicked by the induction of CD3 signaling in thymocytes of recombinase activating gene (RAG)-2-deficient mice (RAG-2(-/-)). Upon anti-CD3epsilon treatment in vivo, p16(INK4a)-expressing RAG-2(-/-) thymocytes were not rescued from apoptosis, nor could they differentiate. Our data demonstrate that expression of p16(INK4a) prevents the pre-TCR-mediated expansion and/or survival of differentiating thymocytes.     

Cellular response to oncogenic ras involves induction of the Cdk4 and Cdk6 inhibitor p15(INK4b)

The cell cycle inhibitor p15(INK4b) is frequently inactivated by homozygous deletion together with p16(INK4a) and p19(ARF) in some types of tumors. Although the tumor suppressor capability of p15(INK4b) is still questioned, it has been found to be specifically inactivated by hypermethylation in hematopoietic malignancies in the absence of p16(INK4a) alterations. Here we show that, in vitro, p15(INK4b) is a strong inhibitor of cellular transformation by Ras. Surprisingly, p15(INK4b) is induced in cultured cells by oncogenic Ras to an extent similar to that of p16(INK4a), and their expression is associated with premature G(1) arrest and senescence. Ras-dependent induction of these two INK4 genes is mediated mainly by the Raf-Mek-Erk pathway. Studies with activated and dominant negative forms of Ras effectors indicate that the Raf-Mek-Erk pathway is essential for induction of both the p15(INK4b) and p16(INK4a) promoters, although other Ras effector pathways can collaborate, giving rise to a stronger response. Our results indicate that p15(INK4b), by itself, is able to stop cell transformation by Ras and other oncogenes such as Rgr (a new oncogene member of the Ral-GDS family, whose action is mediated through Ras). In fact, embryonic fibroblasts isolated from p15(INK4b) knockout mice are susceptible to transformation by the Ras or Rgr oncogene whereas wild-type embryonic fibroblasts are not. Similarly, p15(INK4b)-deficient mouse embryo fibroblasts are more sensitive than wild-type cells to transformation by a combination of the Rgr and E1A oncogenes. The cell cycle inhibitor p15(INK4b) is therefore involved, at least in some cell types, in the tumor suppressor activity triggered after inappropriate oncogenic Ras activation in the cell.     

Peroxisome proliferator-activated receptor gamma ligands, 15-deoxy-Delta12,14-prostaglandin J2, and ciglitazone, induce growth inhibition and cell cycle arrest in hepatic oval cells

There is growing evidence to show that hepatic oval cells contribute to liver regeneration, dysplastic nodule formation, and hepato-carcinogenesis. Peroxisome proliferator-activated receptors (PPARs) and their ligands play an important role in cell growth, inflammatory responses, and liver pathogenesis including fibrosis and cancer. However, little is known about the role of PPARgamma/its ligands in the growth and differentiation of hepatic oval cells. In this study, we found that OC15-5, a rat hepatic oval cell line, expressed PPARgamma at mRNA and protein levels, and a natural ligand for PPARgamma, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), and a synthetic ligand, ciglitazone, inhibited growth of OC15-5 cells by arresting at G1-S in a dose-dependent manner. Apoptosis was also induced in OC15-5 cells by 15d-PGJ2 treatment. In OC15-5 cells treated with 15d-PGJ2, the expression of CDK inhibitor, p27(Kip1), was up-regulated, while that of p21(WAF1/Cip1), p18(INK4C) CDK2, CDK4, and cyclin E was unchanged. In addition, delayed up-regulation of AFP expression was observed in OC15-5 cells after 15d-PGJ2 or ciglitazone treatment. This is the first report to show that the PPARgamma ligand was involved in the growth, cell cycle, and differentiation of hepatic oval cells, raising the possibility that the PPARgamma ligands may regulate liver regeneration and hepato-carcinogenesis.     

SDF-1alpha/CXCR4: a mechanism for hepatic oval cell activation and bone marrow stem cell recruitment to the injured liver of rats

Stromal derived factor-1 alpha (SDF-1alpha) and its receptor CXCR4 have been shown to play a role in the systematic movement of hematopoietic stem cells (HSC) in the fetal and adult stages of hematopoiesis. Under certain physiological conditions liver oval cells can participate in the regeneration of the liver. We have shown that a percentage of oval cells are of hematopoietic origin. Others have shown that bone marrow derived stem cells can participate in liver regeneration as well. In this study we examined the role of SDF-1alpha and its receptor CXCR4 as a possible mechanism for oval cell activation in oval cell aided liver regeneration. In massive liver injury models where oval cell repair is involved hepatocytes up-regulate the expression of SDF-1alpha, a potent chemoattractant for hematopoietic cells. However, when moderate liver injury occurs, proliferation of resident hepatocytes repairs the injury. Under these conditions SDF-1alpha expression is not up-regulated and oval cells are not activated in the liver. In addition, we show that oval cells express CXCR4, the only known receptor for SDF-1alpha. Lastly, in vitro chemotaxis assays demonstrated that oval cells migrate along a SDF-1alpha gradient which suggests that the SDF-1alpha/CXCR4 interaction is a mechanism by which the oval cell compartment could be activated and possibly recruit a second wave of bone marrow stem cells to the injured liver. In conclusion, these experiments begin to shed light on a possible mechanism, which may someday lead to a better understanding of the hepatic and hematopoietic interaction in oval cell aided liver regeneration.     

Liver stem/progenitor cells: their characteristics and regulatory mechanisms

Liver stem cells give rise to both hepatocytes and bile duct epithelial cells also known as cholangiocytes. During liver development hepatoblasts emerge from the foregut endoderm and give rise to both cell types. Colony-forming cells are present in the liver primordium and clonally expanded cells differentiate into either hepatocytes or cholangiocytes depending on culture conditions, showing stem cell characteristics. The growth and differentiation of hepatoblasts are regulated by various extrinsic signals. For example, periportal mesenchymal cells provide a cue for bipotential hepatoblasts to become cholangiocytes, and mesothelial cells covering the parenchyma support the expansion of foetal hepatocytes by producing growth factors. The adult liver has an extraordinary capacity to regenerate, and after 70% hepatectomy the liver recovers its original mass by replication of the remaining hepatocytes without the activation of liver stem cells. However, in certain types of liver injury models, liver stem/progenitor-like cells, known as oval cells in rodents, proliferate around the portal vein, while the roles of such cells in liver regeneration remain a matter of debate. Clonogenic and bipotential cells are also present in the normal adult liver. In this minireview we describe recent studies on liver stem/progenitor cells by focusing on extracellular signals.     

Hepatic oval 'stem' cell in liver regeneration

Hepatic oval cell activation, proliferation, and differentiation has been observed under certain physiological conditions, mainly when the proliferation of existing hepatocytes has been inhibited followed by severe hepatic injury. Hepatic oval cells display a distinct phenotype and have been shown to be a bipotential progenitor of two types of epithelial cells found in the liver, hepatocytes and bile ductular cells. Bone marrow stem cells have recently been shown to be a potential source of the hepatic oval cells and that reconstitution of an injured liver from a purified stem cell population is possible. The focus of this review is on the studies involving the activation, proliferation, and differentiation of these hepatic oval cells and the role that they play in regeneration of the damaged liver. In order to present the potentiality of the hepatic oval cell, an experimental model that involves the inhibition of normal hepatic growth and division as well as severe hepatic injury via chemical or surgical means has been employed. In this model, an as yet undetermined signal or perhaps the lack of regenerative capability in the hepatocytes activates the hepatic oval cell compartment. However, other than understanding a potential origin of these cells and some of the markers that characterize them, it still remains unclear as to how these cells migrate ('home') into the damaged areas and how they begin their differentiation into mature and functioning hepatic cells.     

Lymphocytes support oval cell-dependent liver regeneration

In case of hepatic damage, the liver uses a unique regeneration mechanism through proliferation of hepatocytes. If this process is inhibited, bipotent oval stem cells proliferate and differentiate to hepatocytes and bile ducts, thus restoring liver mass. Although oval cell accumulation in the liver is often associated with inflammatory processes, the role of lymphocytes in oval cell-mediated hepatic regeneration is poorly understood. We treated wild-type and immunodeficient mice with an oval cell-inducing diet: in the absence of T cells (CD3epsilon(-/-) and Rag2(-/-)) there were fewer oval cells, whereas in alymphoid mice (Rag2(-/-)gamma(c)(-/-)) a strongly reduced oval cell response and higher mortality, due to liver failure, was observed. Adoptive transfer of T cells into alymphoid mice protected them from liver failure, but was insufficient to restore the oval cell response. Treatment of Rag2(-/-) mice with an NK cell-depleting Ab resulted in a significantly diminished oval cell response. These genetic experiments point to a major role for NK and T cells in oval cell expansion. In wild-type mice, oval cell proliferation is accompanied by an intrahepatic inflammatory response, characterized by the recruitment of Kupffer, NK, NKT, and T cells. Under these conditions, lymphocytes produce T(H)1 proinflammatory cytokines (IFN-gamma and TNF-alpha) that are mitogenic for oval cells. Our data suggest that T and NK lymphocytes stimulate oval cell expansion by local cytokine secretion. This beneficial cross-talk between the immune system and liver stem cells operates under noninfectious conditions and could promote tissue regeneration following acute liver damage.     

Hepatoblast-like cells populate the adult p53 knockout mouse liver: evidence for a hyperproliferative maturation-arrested stem cell compartment.

Although p53 regulates the cell cycle and apoptosis, gross embryonic development is normal in the p53 knockout (-/-) mouse. In this study, we comprehensively assessed liver development in p53 -/- mice (from embryonic day 15 to adult) for evidence of a cell cycle-induced perturbation in differentiation. Liver cell proliferation in the embryo and newborn is similar in p53 -/- and +/+ mice; in contrast, -/- adult hepatocytes divide at twice the rate of wild types. Developmental expression patterns of liver-specific markers that are up-regulated (e.g., phosphoenolpyruvate carboxykinase and aldolase B) and down-regulated (e.g., alpha-fetoprotein) are similar. Therefore, embryonic and perinatal liver development is normal in the absence of p53. However, the p53 -/- adult liver displays small blast-like cells, the majority being hepatic and some lymphoid. These cells appear in periportal regions and can infiltrate the parenchyma. The hepatic blast-like cells express both mature and immature liver markers, suggesting that differentiation of the liver stem cell compartment is blocked.     

Geroconversion of aged muscle stem cells under regenerative pressure

Regeneration of skeletal muscle relies on a population of quiescent stem cells (satellite cells) and is impaired in very old (geriatric) individuals undergoing sarcopenia. Stem cell function is essential for organismal homeostasis, providing a renewable source of cells to repair damaged tissues. In adult organisms, age-dependent loss-of-function of tissue-specific stem cells is causally related with a decline in regenerative potential. Although environmental manipulations have shown good promise in the reversal of these conditions, recently we demonstrated that muscle stem cell aging is, in fact, a progressive process that results in persistent and irreversible changes in stem cell intrinsic properties. Global gene expression analyses uncovered an induction of p16^INK4a in satellite cells of physiologically aged geriatric and progeric mice that inhibits satellite cell-dependent muscle regeneration. Aged satellite cells lose the repression of the INK4a locus, which switches stem cell reversible quiescence into a pre-senescent state; upon regenerative or proliferative pressure, these cells undergo accelerated senescence (geroconversion), through Rb-mediated repression of E2F target genes. p16^INK4a silencing rejuvenated satellite cells, restoring regeneration in geriatric and progeric muscles. Thus, p16^INK4a/Rb-driven stem cell senescence is causally implicated in the intrinsic defective regeneration of sarcopenic muscle. Here we discuss on how cellular senescence may be a common mechanism of stem cell aging at the organism level and show that induction of p16^INK4a in young muscle stem cells through deletion of the Polycomb complex protein Bmi1 recapitulates the geriatric phenotype.     

Rb蛋白功能缺陷降低肝细胞对TGF-β的敏感性

目的：探讨肝细胞内视网膜母细胞瘤（Rb）基因缺陷对转化生长因子-β（TGF-β）诱导的细胞周期静止的影响。方法：分离并培养原代肝细胞,特异性siRNA腺病毒封闭细胞内Rb基因表达后加入TGF-β诱导,然后MTT法检测细胞生长变化,流式细胞仪检测细胞周期变化,并用Western blot和荧光定量逆转录聚合酶反应（FQ-RT-PCR）法检测细胞中pRb、E2F、c-MYC和p16的表达变化。结果：原代培养肝细胞感染Rb-siRNA重组腺病毒后,正常对照肝细胞抑制率高于Rb基因封闭的肝细胞（69.76%,p<0.01）,流式细胞检测可见对照组TGF-β诱导后处于G1期的肝细胞数明显增多（99.80%,p<0.05）,而在Rb基因封闭组中,TGF-β诱导后各细胞周期分布与未诱导的正常肝细胞基本一致。Western blotting检测可见48h时对照组TGF-β诱导后,E2F和c-MYC蛋白表达减少而p16高表达,Rb基因封闭组TGF-β诱导后c-MYC表达减少而p16表达增高。结论：TGF-β可诱导肝细胞静止于G1期,而Rb基因功能缺陷的肝细胞对TGF-β的诱导敏感性显著降低。     

Loss of CABLES1, a Cyclin-dependent Kinase-interacting Protein that Inhibits Cell Cycle Progression,Results in Germline Expansion at the Expense of Oocyte Quality in Adult Female Mice

Recent studies have shown that cell cycle inhibitors encoded by the Ink4a gene locus constrain the self-renewing activity of adult stem cells of the hematopoietic and nervous systems. Here we report that knockout (KO) of the Cables1 [cyclin-dependent kinase (CDK)-5 and ABL enzyme substrate 1] cell cycle-regulatory gene in mice has minimal to no effect on hematopoietic stem cell (HSC) dynamics. However, female Cables1-null mice exhibit a significant expansion of germ cell (oocyte) numbers throughout adulthood. This is accompanied by a dramatic elevation in the number of atretic immature oocytes within the ovaries and an increase in the incidence of degenerating oocytes retrieved following superovulation of CABLES1-deficient females. These outcomes are not observed in mice lacking p16^INK4a alone or both p16^INK4a and p19^ARF. These data support recent reports that adult female mice can generate new oocytes and follicles but the enhancement of postnatal oogenesis by Cables1 KO appears offset by a reduction in oocyte quality, as reflected by increased elimination of these additional germ cells via apoptosis. This work also reveals cell lineage specificity with respect to the role that specific CDK-interacting proteins play in restraining the activity of adult germline versus somatic stem cells.     

Signaling networks in hepatic oval cell activation

Oval cells are hypothesized to be the progeny of intrahepatic stem cells, also referred to as adult liver stem cells. The mechanisms by which these cells are activated to proliferate and differentiate during liver regeneration is important for the development of new therapies to treat liver disease. Oval cell activation is the first step in progenitor-dependent liver regeneration in response to certain types of injury. This review describes what is currently known about the factors involved in oval cell activation, proliferation, migration, and differentiation.     

Coexpression of Stem Cell Factor andc-kitin Embryonic and Adult Liver

Stem cell factor and its receptorc-kitconstitute an important signal transduction system implicated in survival, proliferation, and differentiation of stem cells in hematopoiesis, gametogenesis, and melanogenesis. In the present study we used both immunocytochemical methods and Western analysis to demonstrate the presence of this cytokine/receptor system in both embryonic and adult rat liver. Stem cell factor was present in the ductular cells around the portal vein during the late embryonic stage of the liver. In the adult liver both bile ducts and bile ductules were positive for stem cell factor andc-kit.When the activation of the liver stem cell compartment was induced by combining administration of acetylaminofluorene and partial hepatectomy, both stem cell factor andc-kitwere expressed in the infiltrating oval cell population, but absent in the newly formed basophilic hepatocytes. Activation of oval cell proliferation following administration ofD-galactosamine also produced a similar but less prominent increase in the level of the stem cell factor. Our data suggest that the stem cell factor/c-kitsignal transduction system is involved in the development of bile ducts and that it may also be an important member of the growth factor/receptor systems associated with the biology of liver stem cells.     

Depletion of HOXA5 inhibits the osteogenic differentiation and proliferation potential of stem cells from the apical papilla

Mesenchymal stem cells (MSCs) are a prospective cell source for tissue regeneration due to their self‐renewal abilities and potential to differentiate into different cell lineages, but the molecular mechanisms of the directed differentiation and proliferation are still unknown. Recently, multiple studies have indicated the crucial role of HOX genes in MSC differentiation and proliferation. However, the role of HOXA5 in MSCs remains unknown. Here, we investigated HOXA5 function in stem cells from the apical papilla (SCAPs). After HOXA5 depletion, the results showed a significant decrease in ALP activity and a weakened mineralization ability of SCAPs. The real‐time RT‐PCR results showed prominently lessened expression of OPN and BSP. The CCK8 and CFSE results displayed inhibited proliferation of SCAPs, and flow cytometry assays revealed arrested cell cycle progression at the S phase. Furthermore, we found that depletion of HOXA5 upregulated p16^INK4A and p18^INK4C and downregulated the Cyclin A. Our research demonstrated that depletion of HOXA5 inhibited osteogenic differentiation and repressed cell proliferation by arresting cell cycle progression at the S phase via p16^INK4A, p18^INK4C, and Cyclin A in SCAPs, indicating that HOXA5 has a significant role in maintaining the proliferation and differentiation potential of dental‐tissue‐derived MSCs.     

Genetic abolishment of hepatocyte proliferation activates hepatic stem cells

Quiescent hepatic stem cells (HSCs) can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1), an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs) by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.     

In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18INK4C

Self-renewal of stem cells is critical for tissue repair and maintenance of organ integrity in most mammalian systems. The relative asymmetry between self-renewal and differentiation in balance with apoptosis determines the size and durability of a stem-cell pool. Regulation of the cell cycle is one of the fundamental mechanisms underlying determination of cell fate. Absence of p21(Cip1/Waf1), a late G1-phase cyclin-dependent kinase inhibitor (CKI), has previously been shown to enable cell-cycle entry of haematopoietic stem cells, but leads to premature exhaustion of the stem cells under conditions of stress. We show here that deletion of an early G1-phase CKI, p18(INK4C), results in strikingly improved long-term engraftment, largely by increasing self-renewing divisions of the primitive cells in murine transplant models. Therefore, different CKIs have highly distinct effects on the kinetics of stem cells, possibly because of their active position in the cell cycle, and p18(INK4C) appears to be a strong inhibitor limiting the potential of stem-cell self-renewal in vivo.     

An Opposite Effect of the CDK Inhibitor,p18INK4c on Embryonic Stem Cells Compared with Tumor and Adult Stem Cells

Self-renewal is a feature common to both adult and embryonic stem (ES) cells, as well as tumor stem cells (TSCs). The cyclin-dependent kinase inhibitor, p18^INK4c, is a known tumor suppressor that can inhibit self-renewal of tumor cells or adult stem cells. Here, we demonstrate an opposite effect of p18 on ES cells in comparison with teratoma cells. Our results unexpectedly showed that overexpression of p18 accelerated the growth of mouse ES cells and embryonic bodies (EB); on the contrary, inhibited the growth of late stage teratoma. Up-regulation of ES cell markers (i.e., Oct4, Nanog, Sox2, and Rex1) were detected in both ES and EB cells, while concomitant down-regulation of various differentiation markers was observed in EB cells. These results demonstrate that p18 has an opposite effect on ES cells as compared with tumor cells and adult stem cells. Mechanistically, expression of CDK4 was significantly increased with overexpression of p18 in ES cells, likely leading to a release of CDK2 from the inhibition by p21 and p27. As a result, self-renewal of ES cells was enhanced. Our current study suggests that targeting p18 in different cell types may yield different outcomes, thereby having implications for therapeutic manipulations of cell cycle machinery in stem cells.     

p18~(INK4C)调控小鼠T细胞的发育及功能

p18INK4C属于细胞周期蛋白激酶抑制剂,其突变或缺失与某些肿瘤的发生密切相关,如T细胞白血病,但目前关于p18调控T细胞发育及功能的研究还鲜有报道,其调控机制仍不明确.本研究利用p18基因敲除(p18KO)小鼠,系统地研究了胸腺中T细胞的早期发育及成熟T细胞的增殖和活化功能,并利用逆转录病毒的方法在Lin?造血干祖细胞上过表达p18,移植4个月后检测其对T细胞的影响.结果表明,p18的缺失对胸腺T细胞的早期发育影响不明显,但随着p18KO小鼠周龄的增加会促进CD4+CD8+双阳性T细胞的数量,此外,p18还通过影响CD3+成熟T细胞的细胞周期进程及IFN-?,GATA3,Tbx21和Foxp3等的表达增强脾脏T细胞的增殖和活化;进一步在造血干祖细胞上过表达p18后会影响T细胞的发育和成熟,进而纠正T细胞在数量上的异常.本研究阐释了p18在T细胞早期发育及后期活化中的调控机制,并证实可通过在干祖细胞水平改变p18的表达进而影响T细胞的分化,这对p18调控T细胞功能异常及参与T细胞白血病的发生提供了新的理论依据和重要的研究价值.