New patterns of nuclear lamina induced by cell fusion

During the eukaryote cell cycle the nuclear envelope displays a series of major morphogenetic changes, the most significant of which include its breakdown and reconstitution as cells move up to, pass through and emerge from division. The three polypeptides, lamins A, B and C, are major components of the nuclear pore complex-lamina fraction of the nuclear envelope and their association with the nuclear membrane or their dispersal in the cytoplasm reflects the existing balance between polymerization and depolymerization in the envelope. We have perturbed the lamina polymerization cycle by means of cell fusion between mitotic and interphase cells, following the redistribution of nuclear lamina protein by means of immunofluorescence techniques. In these heterophasic heterokaryons changes in the distribution of lamina occur as a function of (1) the time elapsed after fusion; (2) the ratio of mitotic to interphase elements in the cell, and (3) the stage in the cell cycle occupied by the interphase partner at the time of fusion. Depolymerization of nuclear lamina occurs most rapidly in cells with high ratios of mitotic to interphase elements, and especially in G1 rather than S-phase nuclei. While lamina depolymerization predominates early after fusion, at later times lamina is deposited around both the original metaphase and interphase nuclear masses and this is associated with the resumption of interphase activity in the form of limited DNA synthesis. These observations lead us to conclude that lamina depolymerization is under positive control mediated by diffusible factors in the cytoplasm of the metaphase partner. Repolymerization is likely to be associated with the inactivation of these factors as the heterokaryons age and, as a result, pass into an interphase-like state.     

RANKL is a downstream mediator for insulin-induced osteoblastic differentiation of vascular smooth muscle cells

Several reports have shown that circulating insulin level is positively correlated with arterial calcification; however, the relationship between insulin and arterial calcification remains controversial and the mechanism involved is still unclear. We used calcifying vascular smooth muscle cells (CVSMCs), a specific subpopulation of vascular smooth muscle cells that could spontaneously express osteoblastic phenotype genes and form calcification nodules, to investigate the effect of insulin on osteoblastic differentiation of CVSMCs and the cell signals involved. Our experiments demonstrated that insulin could promote alkaline phosphatase (ALP) activity, osteocalcin expression and the formation of mineralized nodules in CVSMCs. Suppression of receptor activator of nuclear factor κB ligand (RANKL) with small interfering RNA (siRNA) abolished the insulin-induced ALP activity. Insulin induced the activation of extracellular signal-regulated kinase (ERK)1/2, mitogen-activated protein kinase (MAPK) and RAC-alpha serine/threonine-protein kinase (Akt). Furthermore, pretreatment of human osteoblasts with the ERK1/2 inhibitor PD98059, but not the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, or the Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO), abolished the insulin-induced RANKL secretion and blocked the promoting effect of insulin on ALP activities of CVSMCs. Recombinant RANKL protein recovered the ALP activities decreased by RANKL siRNA in insulin-stimulated CVSMCs. These data demonstrated that insulin could promote osteoblastic differentiation of CVSMCs by increased RANKL expression through ERK1/2 activation, but not PI3K/Akt activation.     

Skeletal muscle cell hypertrophy induced by inhibitors of metalloproteases; myostatin as a potential mediator

Cell growthand differentiation are controlled in many tissues by paracrinefactors, which often require proteolytic processing for activation.Metalloproteases of the metzincin family, such as matrixmetalloproteases and ADAMs, recently have been shown to be involved inthe shedding of growth factors, cytokines, and receptors. In thepresent study, we show that hydroxamate-based inhibitors ofmetalloproteases (HIMPs), such as TAPI and BB-3103, increase the fusionof C₂C₁₂ myoblasts and provoke myotubehypertrophy. HIMPs did not seem to effect hypertrophy via proteins thathave previously been shown to regulate muscle growth in vitro, such asinsulin-like growth factor-I, calcineurin, and tumor necrosis factor-. Instead, the proteolytic maturation of myostatin (growth differentiation factor-8) seemed to be reduced inC₂C₁₂ cells treated with HIMPs, as suggested bythe presence of nonprocessed myostatin precursor only in hypertrophicmyotubes. Myostatin is a known negative regulator of skeletal musclegrowth, belonging to the transforming growth factor-/bonemorphogenetic protein superfamily. These results indicate thatmetalloproteases are involved in the regulation of skeletalmuscle growth and differentiation, that the proteolytic maturation ofmyostatin in C₂C₁₂ cells may be directly orindirectly linked to the activity of some unidentified HIMP-sensitivemetalloproteases, and that the lack of myostatin processing on HIMPtreatment may be a mediator of myotube hypertrophy in this in vitro model.

     

Hsp27 is a mediator of sustained smooth muscle contraction in response to bombesin.

We have identified the low MW 27 kD heat shock protein as a major phosphoprotein constituent of smooth muscle and have investigated its potential role in agonist induced smooth muscle contraction. The neuropeptides bombesin and substance P, which are present in neurons of the anorectal region, induce contraction of isolated smooth muscle cells from this region by activating different intracellular pathways. Substance P-induced contraction is 1,4,5-inositol trisphosphate (IP3)/calmodulin dependent, while contraction induced by bombesin is mediated by a protein kinase C (PKC)-dependent pathway. The sustained contraction induced by bombesin or exogenous PKC was blocked by preincubation of cells with monoclonal antibodies to hsp27, while the transient contraction induced by substance P or IP3 was unaffected by the antibodies. Preincubation with isotype matched control antibodies had no inhibitory effect on contraction induced in response to the agents used. These data support a novel role for hsp27 in the non calmodulin mediated sustained contraction induced by bombesin or PKC.     

Regulation of vascular smooth muscle cell proliferation by nuclear orphan receptor Nur77

Endothelium-derived nitric oxide (NO) is a cytoprotective molecule to prevent endothelial cells (ECs) from apoptosis. CREB-binding protein (CBP) is involved in the apoptotic pathway in several tumor cells, however, little is known whether CBP is associated with apoptosis in ECs and the apoptotic effect of CBP on ECs is regulated by NO. Therefore, the purpose of the present study was to investigate whether silencing CBP expression could affect the sensitivity of ECs toward apoptotic stimuli and determined the role of NO. In this study, we found that when CBP expression was silenced by RNA interference, ECs were more prone to apoptosis under serum deprivation, whereas the apoptosis was not significantly induced in the serum-containing condition. The increased apoptosis is paralleled by a reduction of NO, and the apoptosis was reversed by NO donors, suggesting an important role of NO. Furthermore, CBP silencing decreased NO production by downregulating the endothelial NO synthase (eNOS) expression in a dose-dependent manner. These results indicated that CBP silencing is associated with decreased eNOS expression and NO production, and therefore concomitantly increased the sensitivity of ECs toward apoptosis.     

miR-15b induced by platelet-derived growth factor signaling is required for vascular smooth muscle cell proliferation

The platelet-derived growth factor (PDGF) signaling pathway is essential for inducing a dedifferentiated state of vascular smooth muscle cells (VSMCs). Activation of PDGF inhibits smooth muscle cell (SMC)-specific gene expression and increases the rate of proliferation and migration, leading to dedifferentiation of VSMCs. Recently, microRNAs have been shown to play a critical role in the modulation of the VSMC phenotype in response to extracellular signals. However, little is known about microRNAs regulated by PDGF in VSMCs. Herein, we identify microRNA-15b (miR-15b) as a mediator of VSMC phenotype regulation upon PDGF signaling. We demonstrate that miR-15b is induced by PDGF in pulmonary artery smooth muscle cells and is critical for PDGF-mediated repression of SMC-specific genes. In addition, we show that miR-15b promotes cell proliferation. These results indicate that PDGF signaling regulates SMC-specific gene expression and cell proliferation by modulating the expression of miR-15b to induce a dedifferentiated state in the VSMCs. [BMB Reports 2013; 46(11): 550-554]     

Increased polyploidy in aortic vascular smooth muscle cells during aging is marked by cellular senescence

We previously reported that the frequency of polyploid aortic vascular smooth muscle cells (VSMC) serves as a biomarker of aging. Cellular senescence of somatic cells is another marker of aging that is characterized by the inability to undergo cell division. Here, we examined whether polyploidy is associated with the development of cellular senescence in vivo. Analysis of aortic tissue preparations from young and old Brown Norway rats showed that expression of senescence markers such as p16(INK4a) and senescence-associated beta-galactosidase activity are detected primarily in the old tissues. VSMC from p16(INK4a) knockout and control mice display similar levels of polyploid cells. Intriguingly, senescence markers are expressed in most, but not all, polyploid VSMC. Moreover, the polyploid cells exhibit limited proliferative capacity in comparison to their diploid counterparts. This study is the first to demonstrate in vivo that polyploid VSMC adopt a senescent phenotype.     

Contraction of bovine coronary vascular smooth muscle induced by cocaine is not mediated by norepinephrine

This study was designed to assess the effects of cocaine on coronary arterial smooth muscle and to determine whether previously reported cocaine-induced coronary vasospasm is mediated by substances released from the endothelium or by increased adrenergic receptor stimulation. Concentration-response relationships for cocaine (0.1-300 microM) and norepinephrine (0.1-300 microM) were studied in vitro using 2 mm segments of bovine proximal left anterior descending coronary artery. Each segmental ring was mounted in a 70 ml tissue bath for the measurement of isometric tension. Cocaine (3-300 microM) caused significant, concentration-dependent, increases in developed tension (p less than 0.05). Removal of the endothelium or pretreatment with prazosin (0.1 microM) or propranolol (1 microM) did not significantly alter this action of cocaine. In contrast to cocaine, norepinephrine (10-300 microM) caused significant decreases in developed tension (p less than 0.01). These findings suggest that cocaine-induced contraction of bovine coronary vascular smooth muscle is not mediated by endothelium derived contracting substances or norepinephrine.     

白介素-10抑制TNF-α诱导的血管平滑肌细胞增殖

研究观察了重组人白介素 10 (rhIL 10 )对肿瘤坏死因子 (TNF α)刺激的离体大鼠胸主动脉血管平滑肌细胞增殖、细胞周期及对p4 4 /p4 2丝裂素活化蛋白激酶的影响。实验培养大鼠主动脉血管平滑肌细胞 ,采用MTS/PES法确定血管平滑肌细胞 (vascularsmoothmusclecells,VSMCs)的增殖状态 ;应用流式细胞术测定细胞周期 ;利用p4 4 / 4 2磷酸化抗MAPK抗体的蛋白免疫印迹法测定MAPK蛋白表达。结果显示 :( 1)TNF α处理组与对照组相比 ,TNF α对VSMC增殖具有明显的刺激作用 (P <0 0 5 )。rhIL 10单独应用对VSMCs生长没有影响 (P >0 0 5 )。在TNF α刺激下 ,低至 10ng/ml的rhIL 10可抑制VSMCs的生长 (P <0 0 5 )。流式细胞术测定的结果显示 ,rhIL 10分别可使TNF α作用下的VSMC大部分处于G0 /G1期 ,与对照组相比有明显差异 (P <0 0 1)。 ( 2 )TNF α对p4 4 /p4 2MAPK蛋白表达有显著的增强作用 ,此作用可被rhIL 10抑制。结果提示 ,rhIL 10可抑制TNF α诱导的VSMC增殖及p4 4 /p4 2丝裂素活化蛋白激酶的表达     

Thymic stromal lymphopoietin is a key mediator of breast cancer progression

Inflammation is a double-edged sword that can promote or suppress cancer progression. In this study, we report that thymic stromal lymphopoietin (TSLP), an IL-7-like type 1 inflammatory cytokine that is often associated with the induction of Th2-type allergic responses in the lungs, is also expressed in human and murine cancers. Our studies with murine cancer cells indicate that TSLP plays an essential role in cancer escape, as its inactivation in cancer cells alone was sufficient to almost completely abrogate cancer progression and lung metastasis. The cancer-promoting activity of TSLP primarily required signaling through the TSLP receptor on CD4(+) T cells, promoting Th2-skewed immune responses and production of immunosuppressive factors such as IL-10 and IL-13. Expression of TSLP therefore may be a useful prognostic marker, and its targeting could have therapeutic potential.     

The heavy-chain stoichiometry of smooth muscle myosin is a characteristic of smooth muscle tissues

The stoichiometry of the two heavy chains of myosin in smooth muscle was determined by electrophoresing extracts of native myosin and of dissociated myosin on sodium dodecyl sulfate (SDS) 4%-polyacrylamide gels. The slower migrating heavy chain was 3.6 times more abundant in toad stomach, 2.3 in rabbit myometrium, 2.0 in rat femoral artery, 1.3 in guinea pig ileum, 0.93 in pig trachea and 0.69 in human bronchus, than the more rapidly migrating chain. Both heavy chains were identified as smooth muscle myosin by immunoblotting using antibodies to smooth muscle and non-muscle myosin. The unequal proportion of heavy chains suggested the possibility of native isoforms of myosin comprised of heavy-chain homodimers. To test this, native myosin extracts wer electrophoresed on non-dissociating (pyrophosphate) gels. When each band was individually analysed on SDS-polyacrylamide gel the slowest was found to be filamin and the other bands were myosin in which the relative proportion of the heavy chains was unchanged from that found in the original tissue extracts. Since this is incompatible with either a heterodimeric or a homodimeric arrangement it suggests that pyrophosphate gel electrophoresis is incapable of separating putative isoforms of native myosin.     

Dysfunctional autophagy is a driver of muscle stem cell functional decline with aging

Regeneration of skeletal muscle relies on its resident stem cells, also known as satellite cells, which are normally quiescent. With aging, satellite cell quiescence is lost concomitant with a muscle regenerative decline. Here we demonstrate that autophagy sustains quiescence over time and that its failure with age drives senescence, which accounts for stem cell loss of function. Pharmacological and genetic reestablishment of autophagy restores homeostasis and regenerative functions in geriatric satellite cells, which has relevance for the elderly population.     

LDL-induced impairment of human vascular smooth muscle cells repair function is reversed by HMG-CoA reductase inhibition

Growing human atherosclerotic plaques show a progressive loss of vascular smooth muscle cells (VSMC) becoming soft and vulnerable. Lipid loaded-VSMC show impaired vascular repair function and motility due to changes in cytoskeleton proteins involved in cell-migration. Clinical benefits of statins reducing coronary events have been related to repopulation of vulnerable plaques with VSMC. Here, we investigated whether HMG-CoA reductase inhibition with rosuvastatin can reverse the effects induced by atherogenic concentrations of LDL either in the native (nLDL) form or modified by aggregation (agLDL) on human VSMC motility. Using a model of wound repair, we showed that treatment of human coronary VSMC with rosuvastatin significantly prevented (and reversed) the inhibitory effect of nLDL and agLDL in the repair of the cell depleted areas. In addition, rosuvastatin significantly abolished the agLDL-induced dephosphorylation of myosin regulatory light chain as demonstrated by 2DE-electrophoresis and mass spectrometry. Besides, confocal microscopy showed that rosuvastatin enhances actin-cytoskeleton reorganization during lipid-loaded-VSMC attachment and spreading. The effects of rosuvastatin on actin-cytoskeleton dynamics and cell migration were dependent on ROCK-signalling. Furthermore, rosuvastatin caused a significant increase in RhoA-GTP in the cytosol of VSMC. Taken together, our study demonstrated that inhibition of HMG-CoA reductase restores the migratory capacity and repair function of VSMC that is impaired by native and aggregated LDL. This mechanism may contribute to the stabilization of lipid-rich atherosclerotic plaques afforded by statins.