Translocation, phosphorylation and association of signaling proteins in membrane microdomains, strongly suggest individual imperative roles of PKC1 and RhoA in colonic motility. Phosphorylation of HSP27 is essential for translocation of PKC1 and RhoA during contraction of colonic circular smooth muscle cells (CSMC). Contraction of CSMC is associated with HSP27 phosphorylation, while HSP20 phosphorylation at S16 inhibits contraction. Preliminary results from adult CSMC show: 1) the presence of cav-1 in lipid raft membrane fractions;2) increased acetylcholine (Ach)-induced sequestration of PKC1, phospho-PKC1 (S657) and HSP27 into the lipid rafts and translocation of HSP20 out of the lipid rafts;3) increased association of PKC1 and HSP27 with caveolin 1 (cav-1) in the particulate fraction of CSMC;and 4) silencing of PKC1 (siRNA for PKC1) had an inhibitory effect of both the initial rise and the sustained phase of Ach-induced force generation in 3-dimensional rings bioengineered (3DBR) from human colon CSMC treated with siRNA for PKC1 suggesting a role for PKC1 in force generation and its maintenance. Preliminary data from aged rat CSMC show that isolated lipid raft fractions were depleted of cav-1 and consequently of phospho-PKC1 (S657) concomitant with decreased association of PKC1 and HSP27 with cav-1. Further, adult CSMC transfected with DN cav-1 cDNA, mimicked Ach response of aged CSMC by exhibiting reduced association of PKC1 and HSP27 with cav-1. This correlated with reduced Ach-induced force generation of 3DBR from CSMC of aged rats. These data suggest a crucial role for lipid rafts, cav-1, and HSP27 in normal contractile responses and a reduction in caveolae formation associated with aging CSMC. Reduced caveolae formation could be a critical factor affected by aging and a putative therapeutic target. Preliminary data indicates that ectopic expression of wt-cav-1 reinstated Ach-induced force generation in 3DBR from aged colon. Therefore, we propose to use multilevel functional approaches to study the intricacies of contractile signaling pathways. We will study the spatiotemporal reorganization and relocation of different proteins using live cell imaging, biochemical and molecular biology tools and real time physiological monitoring of contractile response using 3-dimensional rings bioengineered from adult, aged, and stably transfected smooth muscle cells. The data obtained through these approaches will allow us to: 1) Discern the intricate molecular mechanisms responsible for the understanding of the physiology of colonic motility;2) Understand and identify putative disrupted mechanisms affected by aging that contribute to the sluggishness and pathophysiology of contraction of the colon;and 3) Identify and test the possible putative targets to rectify age-related pathophysiology and sluggishness of colonic motility. Public Health Relevance: In summary, we will utilize biochemical, molecular and structural physiological tools we have developed to detect the disruption of normal physiological motor function due to aging and ultimately design therapies to rectify these defects.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK042876-16
Application #
7809658
Study Section
Clinical and Integrative Gastrointestinal Pathobiology Study Section (CIGP)
Program Officer
Hamilton, Frank A
Project Start
1991-06-15
Project End
2011-06-08
Budget Start
2010-05-01
Budget End
2011-06-08
Support Year
16
Fiscal Year
2010
Total Cost
$354,515
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Pediatrics
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Zakhem, Elie; Raghavan, Shreya; Bitar, Khalil N (2014) Neo-innervation of a bioengineered intestinal smooth muscle construct around chitosan scaffold. Biomaterials 35:1882-9
Raghavan, Shreya; Bitar, Khalil N (2014) The influence of extracellular matrix composition on the differentiation of neuronal subtypes in tissue engineered innervated intestinal smooth muscle sheets. Biomaterials 35:7429-40
Raghavan, Shreya; Gilmont, Robert R; Bitar, Khalil N (2013) Neuroglial differentiation of adult enteric neuronal progenitor cells as a function of extracellular matrix composition. Biomaterials 34:6649-58
Bitar, Khalil N; Zakhem, Elie (2013) Tissue engineering and regenerative medicine as applied to the gastrointestinal tract. Curr Opin Biotechnol 24:909-15
Zakhem, Elie; Raghavan, Shreya; Gilmont, Robert R et al. (2012) Chitosan-based scaffolds for the support of smooth muscle constructs in intestinal tissue engineering. Biomaterials 33:4810-7
Somara, Sita; Bashllari, Daniela; Gilmont, Robert R et al. (2011) Real-time dynamic movement of caveolin-1 during smooth muscle contraction of human colon and aged rat colon transfected with caveolin-1 cDNA. Am J Physiol Gastrointest Liver Physiol 300:G1022-32
Bitar, K; Greenwood-Van Meerveld, B; Saad, R et al. (2011) Aging and gastrointestinal neuromuscular function: insights from within and outside the gut. Neurogastroenterol Motil 23:490-501
Hashish, Mohamed; Raghavan, Shreya; Somara, Sita et al. (2010) Surgical implantation of a bioengineered internal anal sphincter. J Pediatr Surg 45:52-8
Somara, Sita; Gilmont, Robert R; Varadarajan, Saranyaraajan et al. (2010) Phosphorylated HSP20 modulates the association of thin-filament binding proteins: caldesmon with tropomyosin in colonic smooth muscle. Am J Physiol Gastrointest Liver Physiol 299:G1164-76
Gilmont, Robert R; Somara, Sita; Bitar, Khalil N (2008) VIP induces PKA-mediated rapid and sustained phosphorylation of HSP20. Biochem Biophys Res Commun 375:552-6

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