Physiological ER Stress Mediates the Differentiation of Fibroblasts

<div><p>Recently, accumulating reports have suggested the importance of endoplasmic reticulum (ER) stress signaling in the differentiation of several tissues and cells, including myoblasts and osteoblasts. Secretory cells are easily subjected to ER stress during maturation of their secreted proteins. Skin fibroblasts produce and release several proteins, such as collagens, matrix metalloproteinases (MMPs), the tissue inhibitors of metalloproteinases (TIMPs) and glycosaminoglycans (GAGs), and the production of these proteins is increased at wound sites. Differentiation of fibroblasts into myofibroblasts is one of the key factors for wound healing and that TGF-β can induce fibroblast differentiation into myofibroblasts, which express α-smooth muscle actin. Well-differentiated myofibroblasts show increased production of collagen and TGF-β, and bring about wound healing. In this study, we examined the effects of ER stress signaling on the differentiation of fibroblasts, which is required for wound healing, using constitutively ER stress-activated primary cultured fibroblasts. The cells expressed positive α-smooth muscle actin signals without TGF-β stimulation compared with control fibroblasts. Gel-contraction assays suggested that ER stress-treated primary fibroblasts caused stronger shrinkage of collagen gels than control cells. These results suggest that ER stress signaling could accelerate the differentiation of fibroblasts to myofibroblasts at injured sites. The present findings may provide important insights for developing therapies to improve wound healing.</p></div

The repeated TM treated fibroblasts showed α-SMA induction.

<p>Rep-TM and Rep-DMSO cells were cultured at basal medium for 12 h (upper panels) and each cells were treated with TGF-β1 after basal medium condition for 48 h. The cells were fixed and immunocytochemistry was performed with anti- α-SMA antibody.</p

Effects of repeated TM stimulation on fibroblasts’ morphology and GRP78/BiP expression.

<p>Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. After this repeated TM or DMSO stimulation, medium was changed to DMEM with 2% horse serum (Basal medium condition) and incubated for 12h to induce differentiation. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. (a) Just after this repeated TM or DMSO stimulation, the cells were observed (upper panels) and stained by anti-Bip antibody (bottom panels). (b) The cells treated with TM (Rep-TM) or DMSO (Rep-DM/Rep-DMSO) cultured in the culture condition medium (C.C.) or in the Basal medium condition for differentiation (M.C.) were collected and lysed. Western blot analysis was performed using an anti-Bip or anti-β-actin primary antibody (upper panels). Quantitative data were obtained by densitometry of the bands. Data are expressed as the mean ± SEM for at least three independent experiments (shown as a ratio of the Rep-DM C.C.). The P value was compared with the control and calculated by Student's T test. (c) Left and middle panels show the cells treated with TM (Rep-TM) or DMSO (Rep-DMSO) cultured at Basal medium condition. Right panel shows the cells treated with TGF-β1 after the incubation at the basal medium condition.</p

Condition setteing for physiological ER stressed fibroblast.

<p>(a) (upper panels) Primary cultured fibroblasts were treated with 1μg/ml TM. The cells were observed each time points. (middle and bottom panels) Primary cultured fibroblasts were transiently treated with 1μg/ml TM for 24 h or 30 min and the medium were changed to the culture medium. The cells were observed each time points after the medium change. (b-d) The effect of each ER stress methods on cell viability was measured by WST-1 assay. (b) 24h or 5 min of transient 2μg/ml TM stimulation, (c) 5 min of repeated 2μg/ml TM stimulation and (d) 1h or 5 min of repeated 1μg/ml TM stimulation were adopted for these assays. Same amount of DMSO were used as controls. The P value was compared with the control and calculated by Student's T test.</p