CONTRACTILE PROTEIN ACCUMULATION IN AIRWAY SMOOTH MUSCLE

哮喘气道重塑的一个特征是过度堆积原本看起来正常的平滑肌。在急性哮喘发作时，这块肌肉的收缩会收缩气道腔。鉴于其在哮喘气道中的过多及其在气流阻塞中的关键作用，了解气道平滑肌如何调节其收缩装置的表达和积聚是至关重要的，这也是我们的目标。我们开发了一种新的细胞培养系统，在该系统中，在长时间(大于7d)的血清剥夺期间，一部分呼吸道肌细胞在结构和功能上成为收缩细胞。该系统的初步研究表明，收缩装置蛋白的积累是在平滑肌基因转录水平上调节的，通过控制mRNA的翻译，并通过每个肌细胞与周围细胞外基质的相互作用来调节。SA编号1：以SM22和平滑肌肌球蛋白重链(SmMHC)为模型基因，确定调节平滑肌收缩装置基因转录的机制。初步研究表明，血清反应因子(SRF)是SM22和smMHC转录的关键调节因子，并使我们检验了以下假设：(I)SM22和smMHC基因的转录活性是通过SRF活性的变化来控制的。(Ii)反过来，SRF活性受两个上游调节因子--Rho家族GTP酶和细胞外信号调节激酶(ERK 1/2)控制，Rho家族GTP酶可增加SRF活性，ERK 1/2可降低SRF活性。(Iii)Rho家族GTP酶通过下游中间产物Rho激酶(p160ROCK-1)激活SRF。确定决定SM22和smMHC蛋白积累的转录后调控机制。SM22和smMHC在编码mRNAs后在我们的系统中积累很长时间，通过控制翻译效率和/或蛋白质分解代谢来调节；其他研究表明，PI 3-K和p70 S6 K反过来调节翻译。因此，我们将检验以下假设：(I)PI 3-激酶的激活是有效的SM22和smMHC mRNA翻译所必需的，但不是充分的。(Ii)S6激酶的激活是有效的SM22和smMHC mRNA翻译所必需的，也是充分的。(3)血清喂养的心肌细胞中SM22和SMMHC蛋白的加速分解也导致了信使核糖核酸和蛋白质的解离。确定层粘连蛋白的分泌和结合在调节气道平滑肌收缩装置堆积中的作用。在初步研究中，缺乏血清的心肌细胞积累了大量的smMHC，也独特地分泌层粘连蛋白并表达细胞表面层粘连蛋白受体。有待检验的假设是：(I)SM22和smMHC的积累需要层粘连蛋白的自分泌和随后的层粘连蛋白特异性表面受体的结合。(Ii)层粘连蛋白-层粘连蛋白受体相互作用刺激IGF的自分泌。(Iii)IGF反过来刺激收缩的呼吸道肌细胞分泌层粘连蛋白，从而形成正向调节循环。(4)IGFS还可刺激气道收缩肌细胞的PI-3-K和S6-K，从而进一步促进收缩装置蛋白的积聚。总而言之，这些研究应该勾勒出收缩装置积累所依赖的三种调节机制。除了它在阐明平滑肌细胞生物学和哮喘气道重塑的一个整体特征方面的基本价值外，这些知识还可能在建议如何减少肥厚的气道平滑肌中的平滑肌收缩装置积聚方面具有很好的实用价值。

One hallmark of asthmatic airway remodeling is an excess accumulation of otherwise normal-appearing smooth muscle. Contraction of this muscle constricts the airway lumen during acute asthma attacks. Given its overabundance in asthmatic airways and its critical role in airflow obstruction, understanding how airway smooth muscle regulates expression and accumulation of its contractile apparatus is of fundamental importance, and is our goal. We have developed a novel cell culture system in which a subset of airway myocytes become structurally and functionally contractile cells during prolonged (greater than 7d) serum deprivation. Preliminary studies in this system indicate that accumulation of contractile apparatus proteins is regulated at the level of smooth muscle gene transcription, through control of mRNA translation, and by interaction of each myocyte with surrounding extracellular matrix. SA number 1: Identify mechanisms that regulate transcription of smooth muscle contractile apparatus genes, using SM22 and smooth muscle myosin heavy chain (smMHC) as model genes. Pilot studies implicate serum response factor (SRF) as a key regulator of SM22 and smMHC transcription, and lead us to test the hypotheses that: (i) Transcriptional activity of SM22 and smMHC genes is controlled through changes in SRF activity. (ii) In turn, SRF activity is controlled by two upstream regulators -- Rho family GTPases, which increase SRF activity; and extracellular signal-regulated kinase (ERK 1/2), which decreases SRF activity. (iii) Rho family GTPases activate SRF through a downstream intermediate, Rho kinase (p160ROCK-1). SA number 2. Identify post-transcriptional regulatory mechanisms that determine SM22 and smMHC protein accumulation. SM22 and smMHC accumulate long after their encoding mRNAs in our system, revealing regulation through control of translation efficiency and/or protein catabolism; additional studies suggest that PI 3-kinase and p70 S6 kinase in turn regulate translation. Therefore, we will test the hypotheses that: (i) Activation of PI 3-kinase is required but not sufficient for efficient SM22 and smMHC mRNA translation. (ii) Activation of S6 kinase is required and sufficient for efficient SM22 and smMHC mRNA translation. (iii) The dissociation in mRNA vs. protein accumulation noted also stems from accelerated catabolism of SM22 and smMHC proteins in serum-fed myocytes. SA number 3. Identify the role of laminin secretion and binding in regulating contractile apparatus accumulation in airway smooth muscle. In pilot studies, serum-deprived myocytes that accumulated abundant smMHC also uniquely secreted laminin and expressed cell surface laminin receptors. Hypotheses to be tested are: (i) Autocrine secretion of laminin and subsequent binding of laminin-specific surface receptors are required for SM22 and smMHC accumulation. (ii) Laminin-laminin receptor interactions stimulate autocrine IGF secretion. (iii) IGFs in turn stimulate laminin secretion by contractile airway myocytes, resulting in a positive regulatory cycle. (iv) IGFs also stimulate PI 3-kinase and S6 kinase in contractile airway myocytes, thereby further promoting contractile apparatus protein accumulation. Together, these studies should delineate three of the regulatory mechanisms on which contractile apparatus accumulation depends. Beyond its basic value in elucidating an integral feature of smooth muscle cell biology and asthmatic airway remodeling, this knowledge may well have practical value in suggesting ways to reduce smooth muscle contractile apparatus accumulation in hypertrophied airway smooth muscle.