Diminished response of Werner's syndrome fibroblasts to growth factors PDGF and FGF

Patients with Werner's syndrome, an autosomal recessive disorder, undergo an accelerated aging process that leads to premature death. Fibroblasts from such patients typically grow poorly in culture. Here it is shown that fibroblasts from a patient with Werner's syndrome have a markedly attenuated mitogenic response to platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF). In contrast, they have a full mitogenic response to fetal bovine serum. Both PDGF binding and receptor numbers per cell are unaltered. The Werner's syndrome cells express high constitutive levels of collagenase in vitro. Although PDGF enhances collagenase expression through increased levels of hybridizable collagenase messenger RNA in normal skin fibroblasts, no induction of collagenase occurs in the Werner's syndrome fibroblasts. Moreover, the failure to respond to this agonist effect of PDGF is not restored by fetal bovine serum. The data suggest that failure of one or more PDGF-mediated pathways in Werner's syndrome cells may contribute to the phenotypic expression of the disorder.     

Vasoconstriction: a new activity for platelet-derived growth factor

Platelet-derived growth factor (PDGF) is a potent mitogen for vascular smooth muscle cells that has been implicated in the pathogenesis of atherosclerosis. The potential role of PDGF in the altered vasoreactivity of atherosclerotic vessels has been studied through an examination of its effects on contractility in the rat aorta. PDGF caused a concentration-dependent contraction of aortic strips and was significantly more potent on a molar basis than the classic vasoconstrictor peptide angiotensin II. Furthermore, PDGF increased the cytosolic free calcium concentration in cultured rat aortic smooth muscle cells. These observations suggest a new biological activity for PDGF that may contribute to the enhanced vasoreactivity of certain atherosclerotic vessels.     

A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF.

Macrophage-like U-937 cells secrete a 22-kilodalton heparin-binding growth factor that is mitogenic for BALB-3T3 fibroblasts and smooth muscle cells, but not endothelial cells. The amino acid sequence predicted from complementary DNA clones indicates that the mitogen is a new member of the epidermal growth factor (EGF) family. This heparin-binding EGF-like growth factor (HB-EGF) binds to EGF receptors on A-431 epidermoid carcinoma cells and smooth muscle cells, but is a far more potent mitogen for smooth muscle cells than is EGF. HB-EGF is also expressed in cultured human macrophages and may be involved in macrophage-mediated cellular proliferation.     

A common PDGF receptor is activated by homodimeric A and B forms of PDGF

The human platelet-derived growth factor (PDGF) receptor complementary DNA was cloned and expressed by transfection of Chinese hamster ovary (CHO) fibroblasts. The ability of CHO cells expressing the human receptor complementary DNA (CHO-HR5) to interact with different recombinant forms of PDGF (AA and BB homodimers) was tested. Both forms of PDGF bind to the transfected receptor, stimulate the receptor tyrosine kinase activity, and elicit a mitogenic response in a manner that was indistinguishable from the responses of Balb/c 3T3 cells to AA and BB forms of PDGF can be attributed to a single type of receptor and show that the AA form, like the BB form, is a true mitogen.     

Expression of a platelet-derived growth factor-like protein in simian sarcoma virus transformed cells

The near identity of the partial amino acid sequence of human platelet-derived growth factor (PDGF) and that predicted for p28sis, the putative transforming protein of the simian sarcoma virus (SSV), suggests expression of a growth factor activity may be central for transformation by SSV. It is now reported that SSV-transformed cells but not control cells contain a growth factor activity that is identical to PDGF in immunoassay, in mitogenic dose response, and in specific mitogenic activity. The protein immunoprecipitated by antiserum to human PDGF has an apparent molecular weight of 20,000, identical to that of p20sis, the putative intracellular degradation product of p28sis. The results support the concept that expression of a PDGF-like molecule, which appears to be the product of the viral-sis gene, is responsible for the abnormal regulation of growth is SSV-transformed cells.     

Interferon inhibits the establishment of competence in Go/S-phase transition

Addition of mouse interferon-alpha/beta (IFN) to confluent, quiescent BALB/c 3T3 (clone A31) mouse fibroblasts resulted in a block or delay in serum-induced activation of the cell cycle. It was necessary to add IFN within 6 hours after serum stimulation to inhibit nuclear labeling with [3H]thymidine. This is consistent with the time required for platelet-derived growth factor (PDGF) to induce cells to become competent to respond to additional growth factors present in platelet-poor plasma. Simultaneous addition of IFN with PDGF inhibited the PDGF-induced synthesis of a 29-kilodalton and a 35-kilodalton protein that normally occurs within 1 hour after PDGF addition. IFN also suppressed the general increase in protein synthesis that occurs by the fifth hour after PDGF addition. These results show that IFN antagonizes the action of PDGF, thereby interfering with the activation of Go cells for G1 traverse and S-phase entry.     

Evidence that the v-sis gene product transforms by interaction with the receptor for platelet-derived growth factor

A scheme for partial purification of biologically active v-sis-coded protein from cells transformed with simian sarcoma virus (SSV) has made possible a functional comparison of the transforming protein with platelet-derived growth factor (PDGF). The SSV-transforming gene product is capable of specifically binding PDGF receptors, stimulating tyrosine phosphorylation of PDGF receptors, and inducing DNA synthesis in quiescent fibroblasts. Each of these activities was specifically inhibited by antibodies to different regions of the v-sis gene product. Moreover, viral infection of a variety of cell types revealed a strict correlation between those cells possessing PDGF receptors and those susceptible to transformation by SSV. These findings provide evidence that SSV-transforming activity is mediated by the interaction of a virus-coded mitogen with PDGF receptors.     

Neuroleukin: a lymphokine product of lectin-stimulated T cells

Neuroleukin is a lymphokine product of lectin-stimulated T cells that induces immunoglobulin secretion by cultured human peripheral blood mononuclear cells. Neuroleukin acts early in the in vitro response that leads to formation of antibody-secreting cells, but continued production of immunoglobulin by differentiated antibody-secreting cells is neuroleukin-independent. Although the factor is not directly mitogenic, cellular proliferation is a late component of the response to neuroleukin. Neuroleukin does not have B-cell growth factor (BCGF) or B-cell differentiation factor (BCDF) activity in defined assays. Neuroleukin-evoked induction of immunoglobulin secretion is both monocyte- and T-cell-dependent.     

Localization of PDGF-B protein in macrophages in all phases of atherogenesis

Lesions of atherosclerosis occur in the innermost layer of the artery wall and consist primarily of proliferated smooth muscle cells surrounded by large amounts of connective tissue, numerous lipid-laden macrophages, and varying numbers of lymphocytes. Growth-regulatory molecules may be involved in intimal accumulation and proliferation of smooth muscle cells responsible for the occlusive lesions of atherosclerosis. Platelet-derived growth factor (PDGF) B-chain protein was found within macrophages in all stages of lesion development in both human and nonhuman primate atherosclerosis. Thus macrophages may play a critical role in the disease by providing PDGF, a potent chemotactic and growth-stimulatory molecule, to the intimal smooth muscle cells.     

Mitogens and oncogenes can block the induction of specific voltage-gated ion channels

The mechanisms underlying the ontogeny of voltage-gated ion channels in muscle are unknown. Whether expression of voltage-gated channels is dependent on mitogen withdrawal and growth arrest, as is generally true for the induction of muscle-specific gene products, was investigated in the BC3H1 muscle cell line by patch-clamp techniques. Differentiated BC3H1 myocytes expressed functional Ca2+ and Na+ channels that correspond to those found in T tubules of skeletal muscle. However, Ca2+ and Na+ channels were first detected after about 5 days of mitogen withdrawal. In order to test whether cellular oncogenes, as surrogates for exogenous growth factors, could prevent the expression of ion channels whose induction was contingent on mitogen withdrawal, BC3H1 cells were modified by stable transfection with oncogene expression vectors. Expression vectors containing v-erbB, or c-myc under the control of the SV40 promoter, delayed but did not prevent the appearance of functional Ca2+ and Na+ channels. In contrast, transfection with a Val12 c-H-ras vector, or cotransfection of c-myc together with v-erbB, suppressed the formation of functional Ca2+ and Na+ channels for greater than or equal to 4 weeks. Potassium channels were affected neither by mitogenic medium nor by transfected oncogenes. Thus, the selective effects of certain oncogenes on ion channel induction corresponded to the suppressive effects of mitogenic medium.     

Induction of glutamine synthetase in embryonic retina: its dependence on cell interactions

A relation between enzyme induction in embryonic cells and cellular organization is indicated by the finding that the levels of glutamine synthetase induced by hydrocortisone in the embryonic neural retina in vitro are dependent on the associations between the retina cells. Intact retina tissue, aggregates of dissociated cells, and cells in monolayer culture showed a decreasing response, in this order, to glutamine synthetase induction. With time of culture, the enzyme activity continued to rise in the intact retina and in cell aggregates, but activity declined in monolayer cultures even though the inducer was continuously present. Dispersed cells cultured in monolayer without the inducer showed after 24 hours a loss of inducibility which could not be reversed by reaggregating such modified cells but could be prevented by maintaining the freshly dispersed cells at a low temperature.     

IL-15过表达对猪骨骼肌细胞成肌分化的影响

【目的】研究肌肉因子IL-15（interleukin 15）对猪骨骼肌成肌细胞增殖与凋亡的影响,为进一步研究IL-15在动物肌肉品质调控和骨骼肌疾病治疗提供依据。【方法】构建IL-15过表达慢病毒载体GV-492-IL-15,体外无菌分离培养猪骨骼肌卫星细胞,诱导分化,并通过免疫荧光染色进行成肌细胞验证。将成肌细胞转染IL-15过表达重组慢病毒载体,试验分别设置空白对照组（Control）、转染阴性对照病毒组（IL-15-）和转染GV-492-IL-15（IL-15+）慢病毒试验组（n=3）。培养72 h后,收集细胞和培养上清液。分别采用实时定量PCR（qRT-PCR）和Western Blot技术分析目的基因和蛋白的表达情况,采用ELISA试剂盒分析培养液中IL-15含量,采用CCK-8试剂盒分析成肌细胞活力,采用流式细胞术分析细胞周期和细胞凋亡,采用Western Blot技术检测与细胞凋亡密切相关的caspase-3蛋白表达水平的变化。【结果】（1）经鉴定后的质粒转染293T细胞,细胞内可观察到明显的绿色荧光,经Western Blot检测,可以观察到20 kD附近处有特征条带;（2）分离培养的猪骨骼肌卫星细胞呈梭形或纺锤形,诱导后可分化为呈管状的成肌细胞。将分化后的成肌细胞,进行α-SMA单克隆抗体免疫荧光染色,视野中90%的细胞呈阳性反应,胞浆染成红色,表明细胞为骨骼肌成肌细胞。（3）转染GV-492-IL-15慢病毒后,与对照细胞组相比,成肌细胞内IL-15 mRNA和蛋白相对表达量均极显著升高（P<0.001）,但培养液中IL-15蛋白水平变化不大（P>0.05）。CCK-8结果显示,过表达IL-15可增强细胞的增殖能力（P<0.05）。与对照组相比,转染GV-492-IL-15慢病毒的细胞早期凋亡率差异不显著（P>0.05）,但细胞晚期凋亡率显著下降（P<0.05）。与对照组相比,转染慢病毒组细胞中caspase-3蛋白有下降的趋势,但差异不显著（P>0.05）。此外,转染IL-15过表达慢病毒可使G1期细胞比例显著下降,S期和G2/M期细胞比例显著升高（P<0.05）。【结论】在正常生理条件下,IL-15是定位在细胞内并发挥作用的,IL-15过表达对猪骨骼肌成肌细胞早期凋亡没有显著影响,但可以抑制其晚期凋亡,并促进细胞增殖。这一研究将为IL-15正向调控猪骨骼肌肌肉品质和治疗相关肌肉疾病提供技术和理论依据。     

山羊MyoG启动子的克隆和活性检测

【目的】肌细胞生成素(myogenin,MyoG) 是生肌调节因子(MRFs)基因家族中唯一能在骨骼肌细胞发育与生长过程中均可表达的调控因子, 在肌肉细胞分化过程中起着中心调控的作用,它正调控着骨骼肌卫星细胞向成熟肌细胞分化的过程,是唯一不可代替的生肌调节因子。MyoG基因在复制、扩增、基因激活、转录、翻译等多级水平上对肌肉发育进行调控。基因转录的起始阶段是机体生长发育因子进行调控的开端,而此阶段调控的实质是通过启动子和上游调控序列的相互作用,调控目的基因的表达。因此克隆MyoG基因的启动子,探讨启动子区域的启动活性,有助于从理论上揭示MyoG基因表达的关键调控位点,同时也有利于揭示肌肉发育调控的相关机理,为治疗人类相关疾病以及改良家畜肉质研究提供实验依据。本研究克隆山羊Myogenin（MyoG）基因的启动子区域,检测其在哺乳动物骨骼肌细胞内的启动活性。【方法】克隆山羊MyoG基因的启动子序列,连入pDsRed2基本骨架构建了以红荧光蛋白基因为标记基因的真核表达载体pDsRed-GoatMyoG(5.3 kb)。表达载体pDsRed-GoatMyoG经酶切和测序鉴定后,分别转染体外培养的绵羊肌卫星细胞、肌管细胞和成纤维细胞,观察红色荧光蛋白表达情况,然后利用实时荧光定量PCR、Western blot和冰冻切片、免疫组化等技术检测细胞转染后标记基因mRNA 和蛋白在体外培养细胞中的的表达活性。pDsRed-GoatMyoG表达载体对小鼠进行肌肉注射,检测GoatMyoG启动子在体内组织中的启动特异性和效率。肌肉注射5 d后,分别取小鼠的注射腿肌肉组织、非注射腿肌肉组织、睾丸组织、肠组织和肝脏组织,通过实时荧光定量 PCR 检测标记基因DsRed在不同组织中的表达情况。【结果】克隆得到的MyoG启动子序列测序正确,载体pDsRed-GoatMyoG经酶切和测序鉴定,证实载体构建成功;转染质粒 pDsRed-GoatMyoG后,肌卫星细胞和肌管细胞在显微镜下均可观察到细胞发红色荧光,成纤维细胞没有红色荧光。通过实时荧光定量PCR检测得出,转基因肌管细胞内GoatMyoG 启动DsRed 基因表达mRNA 的相对量为14.07;通过Western blot技术检测出转基因肌管细胞含有GoatMyoG启动的DsRed蛋白质,在转基因成纤维细胞内没有检测到 DsRed 蛋白质,说明GoatMyoG启动子可在肌肉组织特异性启动外源基因表达。小鼠肌肉注射pDsRed-GoatMyoG质粒后,注射腿肌肉组织内GoatMyoG 启动DsRed 基因转录mRNA 的相对量为212.32,非注射腿肌肉组织内mRNA的相对量为39.76,注射腿肌肉组织和非注射腿肌肉组织内mRNA 的量均是其它组织的1.99倍以上。通过免疫组化技术在注射腿肌肉组织和非注射腿肌肉组织内均可检测到红色荧光蛋白,在睾丸、肠和肝脏内均未检测到DsRed 蛋白。【结论】山羊MyoG启动子可以特异性的在骨骼肌组织驱动外源基因的表达,是一种有效的肌肉特异性启动子。     

Isoform-specific modulation of Na+, K+-ATPase alpha-subunit gene expression in hypertension

Sodium, potassium-adenosine triphosphatase (Na+, K+-ATPase) is hypothesized to be involved in systemic vascular hypertension through its effects on smooth muscle reactivity and myocardial contractility. By means of RNA blot analyses of cardiac, aortic, and skeletal muscle RNAs in two rat hypertensive models, Na+,K+-ATPase alpha-subunit messenger RNA isoforms (alpha 2 and alpha 3) were shown to be deinduced in response to increased intravascular pressure. The changes were observed after 48 hours or more of experimental hypertension. Under these conditions, there is coordinate induction of another alpha isoform (alpha 1) and of beta-subunit messenger RNAs, probably in response to alterations in sodium flux rather than to elevated blood pressure.     

Inhibition of endothelial regeneration by type-beta transforming growth factor from platelets

Damage to the vessel wall is a signal for endothelial migration and replication and for platelet release at the site of injury. Addition of transforming growth factor-beta (TGF-beta) purified from platelets to growing aortic endothelial cells inhibited [3H]thymidine incorporation in a concentration-dependent manner. A transient inhibition of DNA synthesis was also observed in response to wounding; cell migration and replication are inhibited during the first 24 hours after wounding. By 48 hours after wounding both TGF-beta-treated and -untreated cultures showed similar responses. Flow microfluorimetric analysis of cell cycle distribution indicated that after 24 hours of exposure to TGF-beta the cells were blocked from entering S phase, and the fraction of cells in G1 was increased. The inhibition of the initiation of regeneration by TGF-beta could allow time for recruitment of smooth muscle cells into the site of injury by other platelet components.     

Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-alpha, LPS, and IL-1 beta

Human endothelial cells produced a neutrophil chemotactic factor (NCF) upon stimulation with tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), or lipopolysaccharide (LPS). The expression of endothelial cell-derived NCF messenger RNA and biological activity was both time- and concentration-dependent. Maximal NCF mRNA expression occurred at 10 and at 2 nanograms per milliliter for TNF and IL-1 beta, respectively; mRNA expression was first observed 1 hour after stimulation and was maintained for at least 24 hours. In situ hybridization analysis showed that NCF mRNA peaked in treated cells by 24 hours, whereas unstimulated cells were negative. These studies demonstrated that endothelial cells may participate in neutrophil-mediated inflammation by synthesizing a chemotactic factor in response to specific monokines and LPS.