Proliferation modulates intestinal smooth muscle phenotype in vitro and in colitis in vivo

Abstract

Intestinal inflammation causes an increased intestinal wall thickness, in part, due to the proliferation of smooth muscle cells, which impairs the contractile phenotype elsewhere. To study this, cells from the circular muscle layer of the rat colon (CSMC) were isolated and studied, both in primary culture and after extended passage, using quantitative PCR, Western blot analysis, and immunocytochemistry. By 4 days in vitro, both mRNA and protein for the smooth muscle marker proteins α-smooth muscle actin, desmin, and SM22-α were reduced by >50%, and mRNA for cyclin D1 was increased threefold, evidence for modulation to a proliferative phenotype. Continued growth caused significant further decrease in expression, evidence that phenotypic loss in CSMC was proportional to the extent of proliferation. In CSMC isolated at day 2 of trinitrobenzene sulfonic acid-induced colitis, flow cytometry and Western blotting showed that these differentiated markers were reduced in mitotic CSMC, while similar to control in nonmitotic CSMC. By day 35 post-trinitrobenzene sulfonic acid, when inflammation has resolved, CSMC were hypertrophic, but, nonetheless, showed markedly decreased expression of smooth muscle protein markers per cell. In vitro, day 35 CSMC displayed an accelerated loss of phenotype and increased thymidine uptake in response to serum or PDGF-BB. Furthermore, carbachol-induced expression of phospho-AKT (a marker of cholinergic response) was lost from day 35 CSMC in vitro, while retained in control cells. Therefore, proliferation reduces the expression of smooth-muscle-specific markers in CSMC, possibly leading to altered contractility. However, inflammation-induced proliferation in vivo also causes lasting changes that include unexpected priming for an exaggerated response to proliferative stimuli. Identification of the molecular mechanisms of intestinal smooth muscle cell phenotypic modulation will be helpful in reducing the detrimental effects of inflammation.

Cell culture retains contractile phenotype but epigenetically modulates cell-signaling proteins of excitation-contraction coupling in colon smooth muscle cells

“…Despite the persistence of these changes, the contractile response of smooth muscle cells was normal at this time (35). By contrast, expression of contractile proteins continues to deteriorate in the in vitro environment, as they are cultured repeatedly (16). These differences suggest that, within the environment of the intact colon, the epigenetic mechanisms more or less reset after inflammatory injury to ensure normal function.…”

“…Most studies have confirmed the contractile phenotype of cultured gut smooth muscle cells by demonstrating that they express smooth muscle-specific contractile proteins by immunostaining (17,19,27). Some studies found reduced expression of select contractile proteins in colon (17) and vascular (18) smooth muscle cells but did not investigate the alterations in expression of the cell-signaling proteins involved in excitation-contraction coupling; the contractile proteins receive signals from the cascade of cell-signaling proteins activated during excitation-contraction coupling telling them to contract. These signals, initiated by binding of an agonist, such as ACh, to M 3 receptors in gut smooth muscle cells, define the profile of contraction, such as amplitude and duration.…”

“…We investigated whether proliferation of fully differentiated smooth muscle cells in situ also alters expression of the cell-signaling and contractile proteins. We performed these experiments 35 days after TNBS insult, when the inflammatory response has subsided (16). We found that in situ cell proliferation enhanced expression of G q11 and MYPT1 (Fig.…”

“…TNBS inflammation induces cell hyperplasia in colon smooth muscle cells, resulting in their thickening (16,28). We investigated whether proliferation of fully differentiated smooth muscle cells in situ also alters expression of the cell-signaling and contractile proteins.…”

Global Hypoxia-Ischemia Induced Inflammation and Structural Changes in the Preterm Ovine Gut Which Were Not Ameliorated by Mesenchymal Stem Cell Treatment

“…The inflammation-induced thickening of the circular and longitudinal muscle layers could be explained by several mechanisms, including hypertrophy and/or secretion of inflammatory cytokines, which are considered to have mitogenic effects on muscle cells (46,50,62). In line with these reports, muscle thickening after HI in our animal model was accompanied by increased cell numbers and cell proliferation within the muscle layers of the fetal intestine and elevated mRNA levels of IL-1b and IL-17, two proinflammatory cytokines that are known to promote intestinal muscle growth (50).…”