Several previous reports have demonstrated
Several previous reports have demonstrated that epithelial cell migration is important in promoting mucosal repair after damage caused by DSS and nonsteroidal anti-inflammatory drugs , , . Cell migration is coordinated by many factors, including fibronectin and its receptor integrin α5, EGFR, and matrix metalloproteinases , . Fibronectin, an essential component of the extracellular matrix, has several isoforms such as plasma fibronectin and cellular fibronectin; the latter is synthesized by various cells, including fibroblasts, epithelial cells, chondrocytes, synovial cells, and myocytes . Cellular fibronectin has shown to play an important role in the wound healing process by regulating cell adhesion, spreading, proliferation, migration, and apoptosis , , , , , , , . Fibronectin activity is mediated by the interaction with its receptor integrin α5β1 expressed on several types of cells and is closely involved in cell migration , . Fibronectin is also known for its activity in inflammation, as it interacts with integrins α4β1 and α4β7 mostly expressed on leucocytes, and induces lymphocyte migration , , , , . In fact, novel therapeutic MK0752 against integrin α4β1 (natalizumab) and integrin α4β7 (vedolizumab) have positive effects in patients with multiple sclerosis and IBD (Crohn’s disease and ulcerative colitis), respectively, because they inhibit leucocyte vascular adhesion and migration , . Thus, fibronectin can play both positive as well as negative roles in IBD pathogenesis. In the present study, we observed that the treatment with GPR35 agonists promoted YAMC cell migration and wound repair by increasing the expression of fibronectin and integrin α5, which co-localized with GPR35 in the colonic epithelium of mice with DSS-induced colitis treated with a GPR35 agonist. At the same time, MPO activity and the mRNA expression of proinflammatory cytokines in colonic tissue were significantly inhibited by PA administration in DSS-treated mice. Our results suggest that epithelial cell-derived fibronectin, by binding to α5β1, may locally promote wound repair in the epithelium independently of its association with leucocyte-expressed integrins α4β1 and α4β7, although the precise role of GPR35-mediated upregulation of fibronectin in the epithelium is unknown. A previous study showing that fibronectin produced by epithelial cells promoted mucosal healing in DSS-induced colitis  supports our hypothesis. GPR interaction with G proteins such as Gs, Gi, Gq, and G12/13 triggers intracellular signalling pathways, subsequently upregulating second messengers, including cAMP, Ca2+, inositol 1,4,5-triphosphate, and DAG, and activating protein kinases, which results in the induction of various cellular functions , . GPR35 is mainly coupled with Gq and Gi proteins , , . In the present study, PTX and forskolin, but not PLC and PKC inhibitors, abolished GPR35 agonist-induced migration of YAMC cells, suggesting that GPR35 enhanced the migration of colonic epithelial cells by coupling with Gi protein and decreasing cAMP production. It is known that cAMP exerts a suppressive effect on the migration of intestinal epithelial cells , which is consistent with our results. Several previous reports have indicated that GPR35 signalling is mediated by the phosphorylation of mitogen-activated kinases ERK1/2 , . In the present study, we observed that ERK1/2 phosphorylation was induced only at 6h after cell exposure to a GPR35 agonist, and abolished by the treatment with a GPR35 antagonist and forskolin. However, we did not detect ERK1/2 phosphorylation at earlier time points (5min to 3h) after cell exposure to a GPR35 agonist. It is generally considered that the phosphorylation of ERK1/2 occurs promptly after various stimuli; therefore, it is unlikely that GPR35 agonists may increase the expression of fibronectin and integrin α5 via ERK1/2 phosphorylation. On the other hand, several studies have demonstrated that the interaction between fibronectin and integrin α5β1 induces ERK1/2 activation, resulting in the stimulation of cell adhesion, proliferation, and migration , , . These findings and our present results suggest that the activation of GPR35 upregulates the expression of both fibronectin and integrin α5, and then the downstream signalling promotes wound repair in YAMC cell monolayers mediated by ERK1/2 activation (Fig. 8). Further studies are needed to elucidate the detail of the signalling processes underlying wound repair triggered by GPR35.