CONTRACTILE PROTEIN ACCUMULATION IN AIRWAY SMOOTH MUSCLE

哮喘气道重塑的一个标志是原本正常的平滑肌过度积聚。在急性哮喘发作期间，这块肌肉的收缩会使气道腔收缩。 鉴于其在哮喘气道中的过量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累具有根本重要性，也是我们的目标。 我们已经开发了一种新的细胞培养系统，其中一个子集的气道肌细胞成为结构和功能收缩细胞在长期（大于7天）血清剥夺。 在这个系统中的初步研究表明，收缩器蛋白质的积累是在平滑肌基因转录水平，通过控制mRNA的翻译，并通过每个肌细胞与周围的细胞外基质的相互作用进行调节。 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器基因转录的机制。初步研究暗示血清应答因子（SRF）是SM22和smMHC转录的关键调节因子，并引导我们检验以下假设：（i）SM22和smMHC基因的转录活性通过SRF活性的变化来控制。 (ii)反过来，SRF活性由两个上游调节因子控制-Rho家族GTP酶，其增加SRF活性;和细胞外信号调节激酶（ERK 1/2），其降低SRF活性。 (iii)Rho家族GTP酶通过下游中间体Rho激酶（p160ROCK-1）激活SRF。 SA 2号。确定决定SM22和smMHC蛋白积累的转录后调节机制。 SM22和smMHC在我们的系统中编码mRNA后很长时间内积累，揭示了通过控制翻译效率和/或蛋白质催化剂的调节;其他研究表明PI 3-激酶和p70 S6激酶反过来调节翻译。因此，我们将测试以下假设：（i）PI 3-激酶的激活是有效的SM22和smMHC mRNA翻译所必需的，但不足以。 (ii)S6激酶的激活是有效的SM22和smMHC mRNA翻译所必需的并且是足够的。(iii)所述的mRNA与蛋白质积累的解离也源于血清喂养的肌细胞中SM22和smMHC蛋白的加速catenation。 三号冲锋枪。确定层粘连蛋白分泌和结合在调节气道平滑肌收缩器积聚中的作用。 在初步研究中，积累了丰富的smMHC的血清剥夺的肌细胞也独特地分泌层粘连蛋白和表达细胞表面层粘连蛋白受体。 待检验的假设为：（i）层粘连蛋白的自分泌和随后层粘连蛋白特异性表面受体的结合是SM22和smMHC积累所需的。(ii)层粘连蛋白-层粘连蛋白受体相互作用刺激自分泌IGF分泌。(iii)IGFs反过来刺激层粘连蛋白分泌的收缩气道肌细胞，导致一个积极的调节周期。(iv)IGF还刺激收缩气道肌细胞中的PI 3-激酶和S6激酶，从而进一步促进收缩器蛋白质的积累。 总之，这些研究应该描绘三个调节机制，收缩装置的积累依赖。 除了阐明平滑肌细胞生物学和哮喘气道重塑的整体特征的基本价值外，这一知识在提出减少肥大气道平滑肌中平滑肌收缩装置积聚的方法方面可能具有很大的实用价值。

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.