Lakshmi S. Chaturvedi

Repetitive Deformation Activates Focal Adhesion Kinase and ERK Mitogenic Signals in Human Caco-2 Intestinal Epithelial Cells through Src and Rac1

Intestinal epithelial cells are subject to repetitive deformation during peristalsis and villous motility, whereas the mucosa atrophies during sepsis or ileus when such stimuli are abnormal. Such repetitive deformation stimulates intestinal epithelial proliferation via focal adhesion kinase (FAK) and extracellular signal-regulated kinases (ERK). However, the upstream mediators of these effects are unknown. We investigated whether Src and Rac1 mediate deformation-induced FAK and ERK phosphorylation and proliferation in human Caco-2 and rat IEC-6 intestinal epithelial cells. Cells cultured on collagen-I were subjected to an average 10% cyclic strain at 10 cycles/min. Cyclic strain activated Rac1 and induced Rac1 translocation to cell membranes. Mechanical strain also induced rapid sustained phosphorylation of c-Src at Tyr418, Rac1 at Ser71, FAK at Tyr397 and Tyr576, and ERK1/2 at Thr202/Tyr204. The mitogenic effect of cyclic strain was blocked by inhibition of Src (PP2 or short interfering RNA) or Rac1 (NSC23766). Src or Rac1 inhibition also prevented strain-induced FAK phosphorylation at Tyr576 and ERK phosphorylation but not FAK phosphorylation at Tyr397. Reducing FAK using short interfering RNA blocked strain-induced mitogenicity and attenuated ERK phosphorylation but not Src or Rac1 phosphorylation. Src inhibition blocked strain-induced Rac1 phosphorylation, but Rac inhibition did not alter Src phosphorylation. Transfection of a two-tyrosine phosphorylation-deficient FAK mutant Y576F/Y577F prevented activation of cotransfected myc-ERK2 by cyclic strain. Repetitive deformation induced by peristalsis or villus motility may support the gut mucosa by a pathway involving Src, Rac1, FAK, and ERK. This pathway may present important targets for interventions to prevent mucosal atrophy during prolonged ileus or fasting.

Strain-induced Proliferation Requires the Phosphatidylinositol 3-Kinase/AKT/Glycogen Synthase Kinase Pathway

The intestinal epithelium is repetitively deformed by shear, peristalsis, and villous motility. Such repetitive deformation stimulates the proliferation of intestinal epithelial cells on collagen or laminin substrates via ERK, but the upstream mediators of this effect are poorly understood. We hypothesized that the phosphatidylinositol 3-kinase (PI3K)/AKT cascade mediates this mitogenic effect. PI3K, AKT, and glycogen synthase kinase-3β (GSK-3β) were phosphorylated by 10 cycles/min strain at an average 10% deformation, and pharmacologic blockade of these molecules or reduction by small interfering RNA (siRNA) prevented the mitogenic effect of strain in Caco-2 or IEC-6 intestinal epithelial cells. Strain MAPK activation required PI3K but not AKT. AKT isoform-specific siRNA transfection demonstrated that AKT2 but not AKT1 is required for GSK-3β phosphorylation and the strain mitogenic effect. Furthermore, overexpression of AKT1 or an AKT chimera including the PH domain and hinge region of AKT2 and the catalytic domain and C-tail of AKT1 prevented strain activation of GSK-3β, but overexpression of AKT2 or a chimera including the PH domain and hinge region of AKT1 and the catalytic domain and C-tail of AKT2 did not. These data delineate a role for PI3K, AKT2, and GSK-3β in the mitogenic effect of strain. PI3K is required for both ERK and AKT2 activation, whereas AKT2 is sequentially required for GSK-3β. Furthermore, AKT2 specificity requires its catalytic domain and tail region. Manipulating this pathway may prevent mucosal atrophy and maintain the mucosal barrier in conditions such as ileus, sepsis, and prolonged fasting when peristalsis and villous motility are decreased and the mucosal barrier fails.

Repetitive deformation activates Src-independent FAK-dependent ERK motogenic signals in human Caco-2 intestinal epithelial cells.

Repetitive deformation due to villous motility or peristalsis may support the intestinal mucosa, stimulating intestinal epithelial proliferation under normal circumstances and restitution in injured and inflamed mucosa rich in tissue fibronectin. Cyclic strain enhances Caco-2 and IEC-6 intestinal epithelial cell migration across fibronectin via ERK. However, the upstream mediators of ERK activation are unknown. We investigated whether Src and FAK mediate strain-induced ERK phosphorylation and migration in human Caco-2 intestinal epithelial cells on fibronectin. Monolayers on tissue fibronectin-precoated membranes were subjected to an average 10% repetitive deformation at 10 cycles/min. Phosphorylation of Src-Tyr 418, FAK-Tyr 397-Tyr 576-Tyr 925, and ERK were significantly increased by deformation. The stimulation of wound closure by strain was prevented by Src blockade with PP2 (10 micromol/l) or specific short interfering (si)RNA. Src inhibition also prevented strain-induced FAK phosphorylation at Tyr 397 and Tyr 576 but not FAK-Tyr 925 or ERK phosphorylation. Reducing FAK by siRNA inhibited strain-induced ERK phosphorylation. Transfection of NH2-terminal tyrosine phosphorylation-deficient FAK mutants Y397F, Y576F-Y577F, and Y397F-Y576F-Y577F did not prevent the activation of ERK2 by cyclic strain, but a FAK mutant at the COOH terminal (Y925F) prevented the strain-induced activation of ERK2. Although the Y397F-Y576F-Y577F FAK construct exhibited less basal FAK-Tyr 925 phosphorylation under static conditions, it nevertheless exhibited increased FAK-Tyr 925 phosphorylation in response to strain. These results suggest that repetitive deformation stimulates intestinal epithelial motility across fibronectin in a manner that requires both Src activation and a novel Src-independent FAK-Tyr 925-dependent pathway that activates ERK. This pathway may be an important target for interventions to promote mucosal healing in settings of intestinal ileus or fasting.

Role of RhoA and its effectors ROCK and mDia1 in the modulation of deformation-induced FAK, ERK, p38, and MLC motogenic signals in human Caco-2 intestinal epithelial cells

Repetitive deformation enhances intestinal epithelial migration across tissue fibronectin. We evaluated the contribution of RhoA and its effectors Rho-associated kinase (ROK/ROCK) and mammalian diaphanous formins (mDia1) to deformation-induced intestinal epithelial motility across fibronectin and the responsible focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), p38, and myosin light chain (MLC) signaling. We reduced RhoA, ROCK1, ROCK2, and mDia1 by smart-pool double-stranded short-interfering RNAs (siRNA) and pharmacologically inhibited RhoA, ROCK, and FAK in human Caco-2 intestinal epithelial monolayers on fibronectin-coated membranes subjected to 10% repetitive deformation at 10 cycles/min. Migration was measured by wound closure. Stimulation of migration by deformation was prevented by exoenzyme C3, Y27632, or selective RhoA, ROCK1, and ROCK2 or mDia1 siRNAs. RhoA, ROCK inhibition, or RhoA, ROCK1, ROCK2, mDia1, and FAK reduction by siRNA blocked deformation-induced nuclear ERK phosphorylation without preventing ERK phosphorylation in the cytoplasmic protein fraction. Furthermore, RhoA, ROCK inhibition or RhoA, ROCK1, ROCK2, and mDia1 reduction by siRNA also blocked strain-induced FAK-Tyr(925), p38, and MLC phosphorylation. These results suggest that RhoA, ROCK, mDia1, FAK, ERK, p38, and MLC all mediate the stimulation of intestinal epithelial migration by repetitive deformation. This pathway may be an important target for interventions to promote mechanotransduced mucosal healing during inflammation.

Delineating the signals by which repetitive deformation stimulates intestinal epithelial migration across fibronectin

Repetitive strain stimulates intestinal epithelial migration across fibronectin via focal adhesion kinase (FAK), Src, and extracellular signal-related kinase (ERK) although how these signals act and interact remains unclear. We hypothesized that PI3K is central to this pathway. We subjected Caco-2 and intestinal epithelial cell-6 cells to 10 cycles/min deformation on flexible fibronectin-coated membranes, assayed migration by wound closure, and signaling by immunoblots. Strain stimulated PI3K, AKT, glycogen synthase kinase (GSK), and p38 phosphorylation. Blocking each kinase prevented strain stimulation of migration. Blocking PI3K prevented strain-stimulated ERK and p38 phosphorylation. Blocking AKT did not. Downstream, blocking PI3K, AKT, or ERK inhibited strain-induced GSK-Ser9 phosphorylation. Upstream of AKT, reducing FAK or Rac1 by siRNA blocked strain-stimulated AKT phosphorylation, but inhibiting Src by PP2 or siRNA did not. Transfection with FAK point mutants at Tyr397, Tyr576/577, or Tyr925 demonstrated that only FAK925 phosphorylation is required for strain-stimulated AKT phosphorylation. Myosin light chain activation by strain required FAK, Rac1, PI3K, AKT, GSK, and ERK but not Src or p38. Finally, blebbistatin, a nonmuscle myosin II inhibitor, blocked the motogenic effect of strain downstream of myosin light chain. Thus strain stimulates intestinal epithelial migration across fibronectin by a complex pathway including Src, FAK, Rac1, PI3K, AKT, GSK, ERK, p38, myosin light chain, and myosin II.

Propofol's effects on phagocytosis, proliferation, nitrate production, and cytokine secretion in pressure-stimulated microglial cells

BACKGROUND Intracranial hypertension complicates severe traumatic brain injury frequently and might be associated with poor outcomes. Traumatic brain injury induces a neuroinflammatory response by microglial activation and upregulation of proinflammatory cytokines, such as interleukin-1β, tumor necrosis factor alpha, and interleukin-6. To elucidate the effect of increased intracranial pressure on microglial function, we studied the effects of increased extracellular pressure on primary human microglial cell phagocytosis, proliferation, cytokine secretion, and total nitrate production. In addition, because many patients receive propofol during anesthesia or intensive care unit sedation, we evaluated whether propofol alters the effects of pressure. METHODS Human microglial cells were pretreated with (2.5-20 μg/mL) propofol or Intralipid as a vehicle control were incubated at ambient atmospheric pressure or at 15 or 30 mm Hg increased pressure for 2 h for phagocytosis assays or 24 h for proliferation, cytokine secretion, and total nitrate production studies. Phagocytosis was determined by incorporation of intracellular fluorescent latex beads. Tumor necrosis factor alpha, interleukin-1β, and interleukin-6 were assayed by sandwich enzyme-linked immunosorbent assay and total nitrate by Greiss reagent. RESULTS Increased extracellular pressure stimulated phagocytosis versus untreated microglial cells or cells treated with an Intralipid vehicle control. Propofol also stimulated microglial phagocytosis at ambient pressure. Increased pressure, however, decreased phagocytosis in the presence of propofol. Pressure also increased microglial tumor necrosis factor-α and interleukin-1β secretion and propofol pretreatment blocked the pressure-stimulated effect. Interleukin-6 production was not altered either by pressure or by propofol. Pressure also induced total nitrate secretion, and propofol pretreatment decreased basal as well as pressure-induced microglial nitrate production. CONCLUSION Extracellular pressures consistent with increased intracranial pressure after a head injury activate inflammatory signals in human primary microglial cells in vitro, stimulating phagocytosis, proliferation, tumor necrosis factor-α, interleukin-1β, and total nitrate secretion but not affecting interleukin-6. Such inflammatory events may contribute to the worsened prognosis of traumatic brain injury after increased intracranial pressure. Because propofol alleviated these potentially proinflammatory effects, these results suggest that the inflammatory cascade activated by intracranial pressure might be targeted by propofol in patients with increased intracranial pressure after traumatic brain injury.

Strain matrix-dependently dissociates gut epithelial spreading and motility.

BACKGROUND Repetitive deformation enhances intestinal epithelial migration across tissue fibronectin (tFN) via Src but inhibits migration across collagen. Since cell spreading generally precedes motility, we compared the effects of cyclic strain on Caco-2 spreading and migration on tFN, collagen-I, and plasma fibronectin (pFN), and investigated the role of Src in deformation-influenced spreading and migration. MATERIALS AND METHODS Human Caco-2 intestinal epithelial cells on tFN, collagen-I or pFN were subjected to an average 10% strain at 10 cycles/min for 2 h. Src was inhibited with 10muM PP2 or Src was reduced with siRNA. Parallel studies assessed deformation effects on monolayer wound closure. RESULTS Deformation, Src-inhibition or reduction each inhibited spreading on tFN but Src-inhibition or reduction prevented further inhibition of spreading by deformation without preventing further inhibition of motility. Deformation did not alter spreading on collagen-I or pFN, but inhibited wound closure. CONCLUSIONS Although cell spreading generally precedes and parallels motility, repetitive deformation regulates motility independently of spreading. Since deformation activates Src, the ability of Src blockade to mimic strain-associated inhibition of spreading on tFN suggests that this effect occurs by a separate mechanism that may also require basal Src activity. Further delineation of the mechanisms by which strain disparately modulates spreading and motility may permit acceleration of mucosal healing by targeted interventions to separately promote spreading and epithelial motility.

Schlafen 12 Interaction with SerpinB12 and Deubiquitylases Drives Human Enterocyte Differentiation

Background/Aims: Human enterocytic differentiation is altered during development, fasting, adaptation, and bariatric surgery, but its intracellular control remains unclear. We hypothesized that Schlafen 12 (SLFN12) regulates enterocyte differentiation. Methods: We used laser capture dissection of epithelium, qRT-PCR, and immunohistochemistry to evaluate SLFN12 expression in biopsies of control and fasting human duodenal mucosa, and viral overexpression and siRNA to trace the SLFN12 pathway in human Caco-2 and HIEC6 intestinal epithelial cells. Results: Fasting human duodenal mucosa expressed less SLFN12 mRNA and protein, accompanied by decreases in enterocytic markers like sucrase-isomaltase. SLFN12 overexpression increased Caco-2 sucrase-isomaltase promoter activity, mRNA, and protein independently of proliferation, and activated the SLFN12 putative promoter. SLFN12 coprecipitated Serpin B12 (SERPB12). An inactivating SLFN12 point mutation prevented both SERPB12 binding and sucrase-isomaltase induction. SERPB12 overexpression also induced sucrase-isomaltase, while reducing SERPB12 prevented the SLFN12 effect on sucrase-isomaltase. Sucrase-isomaltase induction by both SLFN12 and SERPB12 was attenuated by reducing UCHL5 or USP14, and blocked by reducing both. SERPB12 stimulated USP14 but not UCHL5 activity. SERPB12 coprecipitated USP14 but not UCHL5. Moreover, SLFN12 increased protein levels of the sucrase-isomaltase-promoter-binding transcription factor cdx2 without altering Cdx2 mRNA. This was prevented by reducing UCHL5 and USP14. We further validated this pathway in vitro and in vivo. SLFN12 or SERPB12 overexpression induced sucrase-isomaltase in human non-malignant HIEC-6 enterocytes. Conclusions: SLFN12 regulates human enterocytic differentiation by a pathway involving SERPB12, the deubiquitylases, and Cdx2. This pathway may be targeted to manipulate human enterocytic differentiation in mucosal atrophy, short gut or obesity.

RHOA and Its Effectors Rho-Associated Kinase (ROK/ROCK) and Mammalian Diaphanous (mDia1) Modulate the Deformation-Induced FAK, ERK, p38 and Mlc Motogenic Signals in Human CACO-2 Intestinal Epithelial Cells

G A A b st ra ct s and an increase in HIF-1α mRNA. Five-day repetitive ASA treatment significantly reduced (by 85%) the area of gastric lesions at day 5 as compared with ASA applied once while decreasing MPO activity, MDA content and significantly raised mRNA for HO-1 but these effects were significantly attenuted in clopidogrel co-administered with ASA. Concurrent treatment with pantoprazole and to the lesser extent with ranitidine, significantly inhibited the area of lesions induced by the combination of ASA and clopidogrel and restored gastric adaptation and the accompanying increase in the GBF in rats treated with ASA and clopidogrel. This restoration of ASA adaptation caused by PPI was accompanied by the downregulation of mRNAs for IL-1β and TNF-α and their plasma levels, the significant decrease in MDA content and an increase in the expression of HO-1 mRNA. We conclude that: 1) clopidogrel acts in synergistic manner with ASA to aggravate ASA damage, 2) dual antiplatelet therapy impairs gastric adaptation to ASA due to downregulation of mRNA for antioxidazing enzyme HO-1, the enhancement in lipid peroxidation, neutrophil-induced MPO activity and the overexpression and release of IL-1β and TNF-α, and 3) PPI pantoprazole affords protection against ASAand clopidogrel-induced damage and restores gastric adaptation to this NSAID in the presence of clopidogrel.

W1709 Role of RHOA GTPase and Its Effectors Rho-Coiled Coiled Kinase (ROCK) and Diaphanous (Mdia) in Modulation of Deformation-Induced MAPK Motogenic Signals in Human CACO-2 Intestinal Epithelial Cells

G A A b st ra ct s with a low dose of indomethacin, orally administered rebamipide suppressed celecoxibinduced mucosal apoptosis and lesion production but did not decrease PGE2 levels in the stomach. Rebamipide also suppressed celecoxib-induced increases in [Ca2+]i, the ER stress response, mitochondrial dysfunction and apoptosis In Vitro. Rebamipide did not suppress increases in [Ca2+]i, ER stress response and apoptosis induced by thapsigargin and ionomycin. We also found that rebamipide suppresses the increases in [Ca2+]i and ER stress response induced by an activator of voltage-dependent L-type Ca2+ channels and that another blocker of this channel suppresses celecoxib-induced increases in [Ca2+]i and ER stress response. Conclusions: These results suggest that celecoxib activates voltage-dependent L-type Ca2+ channels and that rebamipide blocks this activation, resulting in suppression of celecoxib-induced apoptosis. Although oral administration of celecoxib produces significant levels of gastric lesions in humans, especially upon long-term treatment or in patients coadministered with low doses of aspirin, it produces few gastric lesions in animals. We consider that celecoxib does not produce acute gastric lesions in animals due to its inability to decrease the gastric PGE2 level and that an accidental decrease in gastric PGE2 level or that induced by a low dose of aspirin causes the observed celecoxib-dependent production of gastric lesions in humans. Therefore, the observation that rebamipide suppresses celecoxibdependent production of gastric lesions in mice pre-administered with a low dose of indomethacin suggests that rebamipide would be clinically effective for the prevention of celecoxib-produced gastric lesions.