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.
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