Integrin alpha subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation.
Integrin alpha subunit ratios, cytoplasmic domains, and growth factor synergy regulate muscle proliferation and differentiation.
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DOI：
10.1083/jcb.133.1.169
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发表时间：
1996-04
期刊：
影响因子：
--
通讯作者：
Horwitz AF
中科院分区：
文献类型：
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作者：
Sastry SK;Lakonishok M;Thomas DA;Muschler J;Horwitz AF
整合素在肌肉分化中的作用通过整合素α亚基在原代鹌鹑骨骼肌中的异位表达来解决，所述原代鹌鹑骨骼肌是一种特别适合于外源基因的有效转染和表达的培养系统。人α 5亚基或鸡α 6亚基的异位表达产生了相反的表型。α 5转染的成肌细胞保持在增殖期，即使在融合培养物中也受到分化抑制。相反，过表达α 6亚基的成肌细胞表现出抑制的增殖和显著的分化。内源性quail α 6表达的反义抑制抑制成肌细胞分化，导致持续增殖。异位α亚基表达的这些作用在很大程度上由胞质结构域介导。嵌合α亚基，α 5ex/6cyto和α 6 ex/5cyto的异位表达，产生与异位α 5或α 6表达所观察到的表型相反的表型。表达α 5ex/6cyto的成肌细胞显示增殖降低，而分化部分恢复。相比之下，α 6 ex/5cyto转染子保持在增殖期，除非允许其融合至少24小时。此外，在保守的GFFKR基序之前和之后，人α 5亚基胞质结构域截短的表达表明该序列在α 5分化调节中很重要。异位α 5和α 6表达也导致对血清生长因子的促有丝分裂作用的对比反应。表达人α 5亚基的成肌细胞仅在不存在血清的情况下分化，而未转染和α 6转染的成肌细胞的分化对血清浓度不敏感。添加单独的外源性生长因子到α 5转染的成肌细胞中会产生与其对未转染细胞的作用不同的独特反应。bFGF或TGF-β均抑制α 5转染的成肌细胞的无血清分化，但不同之处在于bFGF刺激增殖，而TGF-β抑制增殖。胰岛素或TGF-α促进α 5转染的成肌细胞的增殖和分化;然而，胰岛素改变肌管形态。TGF-α或PDGF-BB增强肌肉α-辅肌动蛋白组织成肌原纤维，这在分化的α 5培养物中受损。除TGF-α外，这些生长因子的作用在未转染的成肌细胞中不明显。最后，成肌细胞在无血清条件下的存活仅在bFGF和胰岛素存在下通过异位α 5表达而增强，而TGF-α和TGF-β促进未转染的成肌细胞的存活。我们的观察结果表明：（1）整合素α亚基在调节成肌细胞增殖和分化中的特异性;（2）整合素表达的比例可以影响增殖或分化的决定;（3）α亚基胞质结构域在介导增殖和分化信号中的作用;和（4）增殖、分化、细胞骨架组装和细胞存活的调节关键取决于不同整联蛋白的表达水平和细胞所处的生长因子环境。
The role of integrins in muscle differentiation was addressed by ectopic expression of integrin alpha subunits in primary quail skeletal muscle, a culture system particularly amenable to efficient transfection and expression of exogenous genes. Ectopic expression of either the human alpha5 subunit or the chicken alpha6 subunit produced contrasting phenotypes. The alpha5-transfected myoblasts remain in the proliferative phase and are differentiation inhibited even in confluent cultures. In contrast, myoblasts that overexpress the alpha6 subunit exhibit inhibited proliferation and substantial differentiation. Antisense suppression of endogenous quail alpha6 expression inhibits myoblast differentiation resulting in sustained proliferation. These effects of ectopic alpha subunit expression are mediated, to a large extent, by the cytoplasmic domains. Ectopic expression of chimeric alpha subunits, alpha5ex/6cyto and alpha6ex/5cyto, produced phenotypes opposite to those observed with ectopic alpha5 or alpha6 expression. Myoblasts that express alpha5ex/6cyto show decreased proliferation while differentiation is partially restored. In contrast, the alpha6ex/5cyto transfectants remain in the proliferative phase unless allowed to become confluent for at least 24 h. Furthermore, expression of human alpha5 subunit cytoplasmic domain truncations, before and after the conserved GFFKR motif, shows that this sequence is important in alpha5 regulation of differentiation. Ectopic alpha5 and alpha6 expression also results in contrasting responses to the mitogenic effects of serum growth factors. Myoblasts expressing the human alpha5 subunit differentiate only in the absence of serum while differentiation of untransfected and alpha6-transfected myoblasts is insensitive to serum concentration. Addition of individual, exogenous growth factors to alpha5-transfected myoblasts results in unique responses that differ from their effects on untransfected cells. Both bFGF or TGFbeta inhibit the serum-free differentiation of alpha5- transfected myoblasts, but differ in that bFGF stimulates proliferation whereas TGF-beta inhibits it. Insulin or TGF-alpha promote proliferation and differentiation of alpha5-transfected myoblasts; however, insulin alters myotube morphology. TGF-alpha or PDGF-BB enhance muscle alpha-actinin organization into myofibrils, which is impaired in differentiated alpha5 cultures. With the exception of TGF- alpha, these growth factor effects are not apparent in untransfected myoblasts. Finally, myoblast survival under serum-free conditions is enhanced by ectopic alpha5 expression only in the presence of bFGF and insulin while TGF-alpha and TGF-beta promote survival of untransfected myoblasts. Our observations demonstrate (1) a specificity for integrin alpha subunits in regulating myoblast proliferation and differentiation; (2) that the ratio of integrin expression can affect the decision to proliferate or differentiate; (3) a role for the alpha subunit cytoplasmic domain in mediating proliferative and differentiative signals; and (4) the regulation of proliferation, differentiation, cytoskeletal assembly, and cell survival depend critically on the expression levels of different integrins and the growth factor environment in which the cells reside.