Dicer-dependent pathways regulate chondrocyte proliferation and differentiation

Small noncoding RNAs, microRNAs (miRNAs), bind to messenger RNAs through base pairing to suppress gene expression. Despite accumulating evidence that miRNAs play critical roles in various biological processes across diverse organisms, their roles in mammalian skeletal development have not been demonstrated. Here, we show that Dicer, an essential component for biogenesis of miRNAs, is essential for normal skeletal development. Dicer-null growth plates show a progressive reduction in the proliferating pool of chondrocytes, leading to severe skeletal growth defects and premature death of mice. The reduction of proliferating chondrocytes in Dicer-null growth plates is caused by two distinct mechanisms: decreased chondrocyte proliferation and accelerated differentiation into postmitotic hypertrophic chondrocytes. These defects appear to be caused by mechanisms downstream or independent of the Ihh-PTHrP signaling pathway, a pivotal signaling system that regulates chondrocyte proliferation and differentiation. Microarray analysis of Dicer-null chondrocytes showed limited expression changes in miRNA-target genes, suggesting that, in the majority of cases, chondrocytic miRNAs do not directly regulate target RNA abundance. Our results demonstrate the critical role of the Dicer-dependent pathway in the regulation of chondrocyte proliferation and differentiation during skeletal development.     

Receptor tyrosine kinase-like orphan receptor 2 (ROR2) and Indian hedgehog regulate digit outgrowth mediated by the phalanx-forming region

Elongation of the digit rays resulting in the formation of a defined number of phalanges is a process poorly understood in mammals, whereas in the chicken distal mesenchymal bone morphogenetic protein (BMP) signaling in the so-called phalanx-forming region (PFR) or digit crescent (DC) seems to be involved. The human brachydactylies (BDs) are inheritable conditions characterized by variable degrees of digit shortening, thus providing an ideal model to analyze the development and elongation of phalanges. We used a mouse model for BDB1 (Ror2^W749X/W749X) lacking middle phalanges and show that a signaling center corresponding to the chick PFR exists in the mouse, which is diminished in BDB1 mice. This resulted in a strongly impaired elongation of the digit condensations due to reduced chondrogenic commitment of undifferentiated distal mesenchymal cells. We further show that a similar BMP-based mechanism accounts for digit shortening in a mouse model for the closely related condition BDA1 (Ihh^E95K/E95K), altogether indicating the functional significance of the PFR in mammals. Genetic interaction experiments as well as pathway analysis in BDB1 mice suggest that Indian hedgehog and WNT/β-catenin signaling, which we show is inhibited by receptor tyrosine kinase-like orphan receptor 2 (ROR2) in distal limb mesenchyme, are acting upstream of BMP signaling in the PFR.     

Progelatinase A is produced and activated by rat hepatic stellate cells and promotes their proliferation.

Activated hepatic stellate cells (HSCs) are a potential source of gelatinase A, which accumulates in fibrotic livers. Progelatinase A activation requires its binding to a complex of membrane-type matrix metalloproteinase (MT-MMP) and tissue inhibitor of metalloproteinases (TIMP)-2. These studies examine gelatinase A, MT1-MMP, and TIMP-2 synthesis by HSCs during activation in vitro and the potential role of gelatinase A in promoting HSC proliferation. Gelatinase A, MT1-MMP, and TIMP-2 messenger RNA (mRNA) were all upregulated in HSCs activated on plastic over 5 to 14 days. Gelatinase A expression was maximal at 7 days of culture, coinciding with the peak of HSC proliferation and the onset of procollagen I and alpha-smooth muscle actin (alpha-SMA) mRNA expression. Active forms of gelatinase A of 62 kd and 66 kd were secreted by activated HSCs and reached a maximum of 12.1% of total enzyme in 14-day culture supernatants. Treatment of HSCs with concanavalin A (con A) induced activation of MT1-MMP and enhanced secretion of activated gelatinase A, which reached a maximum of 44.4% of the total enzyme secreted into culture supernatants using 30 microgram/mL con A. [(14)C]-gelatin degradation assays confirmed the presence of gelatinolytic activity in activated HSC supernatants, which reached a maximum level at 7 days of culture. Antisense oligonucleotide inhibition of endogenous progelatinase A production, or the MMP inhibitor 1,10-phenanthroline inhibited (3)H-thymidine incorporation into HSC DNA by greater than 50%. We conclude that HSCs produce progelatinase A during activation in vitro and activate this enzyme coincident with MT1-MMP and TIMP-2 synthesis. Gelatinase A activity is required for maximal proliferation of HSCs in vitro suggesting this metalloproteinase is an autocrine proliferation factor for HSCs.     

Wnt5a cooperates with canonical Wnts to generate midbrain dopaminergic neurons in vivo and in stem cells

Wnts are a family of secreted proteins that regulate multiple steps of neural development and stem cell differentiation. Two of them, Wnt1 and Wnt5a, activate distinct branches of Wnt signaling and individually regulate different aspects of midbrain dopaminergic (DA) neuron development. However, several of their functions and interactions remain to be elucidated. Here, we report that loss of Wnt1 results in loss of Lmx1a and Ngn2 expression, as well as agenesis of DA neurons in the midbrain floor plate. Remarkably, a few ectopic DA neurons still emerge in the basal plate of Wnt1^−/− mice, where Lmx1a is ectopically expressed. These results indicate that Wnt1 orchestrates DA specification and neurogenesis in vivo. Analysis of Wnt1^−/−;Wnt5a^−/− mice revealed a greater loss of Nurr1⁺ cells and DA neurons than in single mutants, indicating that Wnt1 and Wnt5a interact genetically and cooperate to promote midbrain DA neuron development in vivo. Our results unravel a functional interaction between Wnt1 and Wnt5a resulting in enhanced DA neurogenesis. Taking advantage of these findings, we have developed an application of Wnts to improve the generation of midbrain DA neurons from neural and embryonic stem cells. We thus show that coordinated Wnt actions promote DA neuron development in vivo and in stem cells and suggest that coordinated Wnt administration can be used to improve DA differentiation of stem cells and the development of stem cell-based therapies for Parkinson’s disease.     

雷奈酸锶对大鼠骨髓间充质干细胞增生和成脂分化的影响

目的从细胞和基因水平研究不同浓度雷奈酸锶对大鼠骨髓间充质干细胞成脂分化的影响。方法分离培养并鉴定大鼠骨髓间充质干细胞。以不同浓度雷奈酸锶（0、0．05、0．1、0．5、1．0和5．0mmol／L）刺激骨髓问充质干细胞,测定其增生活性。在成脂诱导条件下,加入不同浓度雷奈酸锶（0、1．0、5．0mmol／L）,倒置显微镜下检测油红染色并进行成脂相关基因检测。结果在非诱导条件下,浓度为1．0和5．0mmol／L的雷奈酸锶对细胞增生具有明显促进作用。在成脂诱导6及9d后,1．0和5．0mmol／L雷奈酸锶均对细胞成脂分化具有抑制作用,表现为细胞内脂滴缩小,油红染色阳性细胞数量减低,成脂分化关键基因PPAR-γ／EBP—β、aP-2以及Glut-4的表达量降低。结论在体外雷奈酸锶具有促进骨髓间充质干细胞增生作用,并抑制其向脂肪细胞分化,这可能与其具有促进前体干细胞成骨分化并维持成骨与成脂分化平衡作用有关。     

Geometric cues for directing the differentiation of mesenchymal stem cells

Significant efforts have been directed to understanding the factors that influence the lineage commitment of stem cells. This paper demonstrates that cell shape, independent of soluble factors, has a strong influence on the differentiation of human mesenchymal stem cells (MSCs) from bone marrow. When exposed to competing soluble differentiation signals, cells cultured in rectangles with increasing aspect ratio and in shapes with pentagonal symmetry but with different subcellular curvature—and with each occupying the same area—display different adipogenesis and osteogenesis profiles. The results reveal that geometric features that increase actomyosin contractility promote osteogenesis and are consistent with in vivo characteristics of the microenvironment of the differentiated cells. Cytoskeletal-disrupting pharmacological agents modulate shape-based trends in lineage commitment verifying the critical role of focal adhesion and myosin-generated contractility during differentiation. Microarray analysis and pathway inhibition studies suggest that contractile cells promote osteogenesis by enhancing c-Jun N-terminal kinase (JNK) and extracellular related kinase (ERK1/2) activation in conjunction with elevated wingless-type (Wnt) signaling. Taken together, this work points to the role that geometric shape cues can play in orchestrating the mechanochemical signals and paracrine/autocrine factors that can direct MSCs to appropriate fates.     

Bile salts regulate proliferation and apoptosis of liver cells by modulating the IGF1 system

BACKGROUND: In different cell types, the insulin-like growth factor 1 and its receptor modulate growth, apoptosis and damage repair in cooperation with estrogen receptors. AIM: To evaluate the involvement of the insulin-like growth factor 1 system and estrogen receptors in bile salts modulation of apoptosis/proliferation of hepatocytes and cholangiocytes. Primary cultures of rat hepatocytes and cholangiocytes were exposed to glycochenodeoxycholate or tauro-CDC in the presence or absence of insulin-like growth factor 1 receptor blocking antibody (alphaIR3), small interfering RNA for insulin-like growth factor 1, 17beta-estradiol or estrogen receptor antagonist (ICI 182,780). Proliferation was evaluated by proliferating cell nuclear antigen Western blot and apoptosis by measuring caspase-3 activity or annexin-V. RESULTS: In hepatocytes, the insulin-like growth factor 1 receptor blocker enhanced glycochenodeoxycholate-induced apoptosis and caused tauro-CDC to promote apoptosis. 17Beta-estradiol or the estrogen receptor antagonist (ICI 182,780) did not influence the apoptotic effect of glycochenodeoxycholate. In cholangiocytes, both glycochenodeoxycholate and tauro-CDC induced proliferation at 100microM, while they induced apoptosis at 1mM with a more pronounced effect of glycochenodeoxycholate. Apoptosis induced by 1mM glycochenodeoxycholate or tauro-CDC in cholangiocytes was enhanced by blocking insulin-like growth factor 1 receptor or by silencing insulin-like growth factor 1. 17Beta-estradiol counteracts glycochenodeoxycholate-induced cholangiocyte apoptosis by enhancing insulin-like growth factor 1 secretion and activating the insulin-like growth factor 1 system. CONCLUSIONS: Modulation of the IGF1 system could represent a potential strategy for the management of bile salts-induced liver injury.     

On the mechanism by which vascular endothelial cells regulate their oxygen consumption

Two enzymes, soluble guanylyl cyclase and cytochrome c oxidase, have been shown to be exquisitely sensitive to nitric oxide (NO) at low physiological concentrations. Activation of the soluble guanylyl cyclase by endogenous NO and the consequent increase in the second messenger cyclic GMP are now known to control a variety of biological functions. Cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, is inhibited by NO. However, it is not clear whether NO produced by the constitutive NO synthase interacts with cytochrome c oxidase, nor is it known what the biological consequences of such an interaction might be. We now show that NO generated by vascular endothelial cells under basal and stimulated conditions modulates the respiration of these cells in response to acute changes in oxygen concentration. This action occurs at the cytochrome c oxidase and depends on influx of calcium. Thus, NO plays a physiological role in adjusting the capacity of this enzyme to use oxygen, allowing endothelial cells to adapt to acute changes in their environment.     

Selective differentiation and proliferation of hematopoietic cells induced by recombinant human interleukins.

Effects of recombinant human interleukins on hematopoiesis were explored by using suspension cultures of mononuclear cells of human umbilical-cord blood and bone marrow. The results showed that interleukin 5 induced the selective differentiation and proliferation of eosinophils. After 3 weeks in culture with interleukin 5, essentially all nonadherent cells in both bone marrow and cord blood cell cultures became eosinophilic myelocytes. Culture of the same cells with interleukin 4 resulted in the selective growth of OKT3+ lymphocytes. However, OKT3+ cells did not develop if the bone marrow cells were depleted of OKT3+/OKT11+ cells prior to the culture, indicating that interleukin 4 induced the proliferation of a subpopulation of resting T cells present in cord blood and bone marrow cell preparations. In suspension cultures of bone marrow cells and cord blood cells grown in the presence of interleukin 3, basophilic, eosinophilic, and neutrophilic myelocytes and macrophages developed within 2 weeks. By 3 weeks, however, the majority of nonadherent cells became eosinophilic myelocytes. In contrast to mouse bone marrow cell cultures, neither interleukin 3 nor a combination of interleukins 3 and 4 induced the differentiation of mast cells in human bone marrow or cord blood cell cultures.     

Soluble factor(s) produced by human bone marrow stroma increase cytokine-induced proliferation and maturation of primitive hematopoietic progenitors while preventing their terminal differentiation

We have recently shown that conservation and differentiation of primitive human hematopoietic progenitors in in vitro long-term bone marrow cultures (LTBMC) occurs to a greater extent when hematopoietic cells are grown separated from the stromal layer than when grown in direct contact with the stroma. This finding suggests that hematopoiesis may depend mainly on soluble factors produced by the stroma. To define these soluble factors, we examine here whether a combination of defined early-acting cytokines can replace soluble stroma-derived biologic activities that induce conservation and differentiation of primitive progenitors. Normal human Lineage- /CD34+/HLA-DR- cells (DR-) were cultured either in the absence of a stromal layer ("stroma-free") or in a culture system in which DR- cells were separated from the stromal layer by a microporous membrane ("stroma-noncontact"). Both culture systems were supplemented three times per week with or without cytokines. These studies show that culture of DR- cells for 5 weeks in a "stroma-free" culture supplemented with a combination of four early acting cytokines (Interleukin-3 [IL-3], stem cell factor [SCF], leukemia-inhibitory factor [LIF], and granulocyte colony-stimulating factor [G-CSF]) results in a similar cell expansion as when DR- cells are cultured in "stroma-noncontact" cultures supplemented with the same cytokines. However, generation of committed progenitors and conservation of the more primitive long-term bone marrow culture initiating cells (LTBMC- IC) was far superior in "stroma-noncontact" cultures supplemented with or without IL-3 than in "stroma-free" cultures supplemented with IL-3 alone or a combination of IL-3, LIF, G-CSF, and SCF. These studies indicate that human BM stroma produces soluble factors that can either alone or in synergy with defined cytokines (1) conserve primitive LTBMC- IC, (2) induce early differentiation of a fraction of the primitive progenitors, and (3) prevent their terminal differentiation. We show here that these stroma-derived factors are not likely to be the known early acting cytokines IL-3, SCF, LIF, or G-CSF. Characterization of the stroma-derived factor(s) may have important implications for clinically relevant studies, such as in vitro stem cell expansion in cancer treatment and gene therapy.     

扶正化瘀方对骨髓细胞在纤维化肝脏中迁移与分化的干预作用

[目的]探究扶正化瘀方(Fuzheng Huayu recipe,FZHY)对骨髓细胞在肝纤维化形成中向肝脏迁移与分化的干预作用.[方法]以绿色荧光蛋白(green fluorescent protein,GFP)标记的供体ICR小鼠的骨髓细胞移植入受体ICR小鼠,8周后进行受体外周血GFP鉴定,并分为移植后对照组与移植后模型组,移植后模型组予二甲基哑硝胺(Dimethylnitrosamine,DMN)诱导肝纤维化,4周后移植后模型组进一步分为移植+DMN组、移植+DMN+肝细胞生长因子(hepatocyte growth-promotting factors,pHGF)组与移植+DMN+ FZHY组,pHGF与FZHY分别给药8周后,免疫荧光检测肝组织的GFP、白蛋白(Albumin,Alb)、α平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)及CD32的表达.[结果]移植8周末受体外周血涂片可见大量GFP;给药12周末,移植+DMN+ FZHY组纤维间隔与肝实质区均可见GFP,实质区可见Alb,未见GFP+/Alb+,纤维间隔内可见GFP+/α-SMA+,但与移植+ DMN组相比明显减少,窦状隙可见CD32+,可见明显GFP+/CD32+.[结论]在本实验建立的骨髓细胞移植后受体肝纤维化形成的体内微环境下,FZHY给药后,骨髓细胞有向肝实质区迁移的趋向,未能向肝细胞分化,可逆转其向肌成纤维细胞的分化失衡,可部分转分化为肝窦内皮细胞.     

肌细胞增强子2A基因突变对血管内皮细胞增殖的影响

目的探讨肌细胞增强子(MEF)2A基因突变对血管内皮细胞增殖的影响。方法采用人脐静脉内皮细胞,分别转染pcDNA3.0-GFP质粒(对照组)、野生型MEF2A质粒(野生型组,WT组)、21碱基缺失MEF2A质粒(Δ21突变组,Δ21组)和MEF2A siRNA片段(siRNA组)。应用细胞免疫荧光法检测MEF2A蛋白定位,MTT法测定内皮细胞增殖情况。结果绿色荧光蛋白结果与Flag抗体检测显示MEF2A基因成功转染,内皮细胞转染效率可达70%。与WT组比较,Δ21组MEF2A突变蛋白表达水平无显著变化,但其定位由核内转变为胞浆,siRNA组则使MEF2A蛋白表达下降80%。与WT组MEF2A转染相比,Δ21组和siRNA组脐静脉内皮细胞增殖率均显著下降(36.2%和63.6%,均为P0.01)。结论 MEF2A基因可调节内皮细胞的增殖,其作用机制可能与MEF2A在细胞核内的定位相关。     

Regulation of rat granulosa cell differentiation by extracellular matrix produced by bovine corneal endothelial cells

The effect of bovine corneal extracellular matrix (ECM) on gonadotropin-primed rat granulosa cells in vitro was studied by examining the following parameters: 1) rate of cell attachment to culture dishes; 2) modulation of cell morphology; 3) specific binding of [125I]human(h)CG to LH/hCG receptors; 4) cAMP response to hCG stimulation; and 5) basal and hCG stimulated progesterone production. Attachment of cells to culture dishes occurred significantly earlier on ECM, as compared with uncoated dishes (6 h vs. 24 h). Cells grown on ECM were epitheloid and organized in multilayer aggregates, closely resembling their organization in the intact wall of the ovarian follicle. In contrast, cultures on uncoated dishes grew as a monolayer of markedly flattened cells. A 2-fold increase in number of LH/hCG receptors occurred on ECM within 48 h, probably due to de novo synthesis. Scatchard analysis revealed no change in hormone affinity to the receptor during the culture period [association constant (Ka) = 2.5 X 10(10)M-1 for hCG]. Cells grown on ECM had a parallel increase in cAMP responsiveness to hCG stimulation. Cells grown in serum-free medium on ECM-coated dishes preserved only 50% of LH/hCG receptors and cAMP responsiveness after 48 h. Cells cultured on ECM showed a marked elevation in progesterone production even in the absence of gonadotropin stimulation, whereas cells grown on uncoated dishes almost completely lost their ability to produce progesterone both in the presence and absence of hCG. These results indicate that ECM plays a substantial role in the maintenance and further propagation of granulosa cell differentiation in vitro.     

Type I interferons produced by dendritic cells promote their phenotypic and functional activation

Resting dendritic cells (DCs) are resident in most tissues and can be activated by environmental stimuli to mature into potent antigen-presenting cells. One important stimulus for DC activation is infection; DCs can be triggered through receptors that recognize microbial components directly or by contact with infection-induced cytokines. We show here that murine DCs undergo phenotypic maturation upon exposure to type I interferons (type I IFNs) in vivo or in vitro. Moreover, DCs either derived from bone marrow cells in vitro or isolated from the spleens of normal animals express IFN-alpha and IFN-beta, suggesting that type I IFNs can act in an autocrine manner to activate DCs. Consistent with this idea, the ability to respond to type I IFN was required for the generation of fully activated DCs from bone marrow precursors, as DCs derived from the bone marrow of mice lacking a functional receptor for type I IFN had reduced expression of costimulatory and adhesion molecules and a diminished ability to stimulate naive T-cell proliferation compared with DCs derived from control bone marrow. Furthermore, the addition of neutralizing anti-IFN-alpha/beta antibody to purified splenic DCs in vitro partially blocked the "spontaneous" activation of these cells, inhibiting the up-regulation of costimulatory molecules, secretion of IFN-gamma, and T-cell stimulatory activity. These results show that DCs both secrete and respond to type I IFN, identifying type I interferons as autocrine DC activators.