| [Chen B, Li J, Fellows GF, Sun Z, Wang R. Maintaining human fetal pancreatic stellate cells function and proliferation require β1 integrin and collagen I matrix interaction. Oncotarget,2015,6(16):14045-14059. Impact factor 6.359]Pancreatic stellate cells (PaSCs) are found in peri-acinar, peri-ductal and peri-vascular regions of the rodent and human pancreas, and regulate extracellular matrix (ECM) turnover and maintain the integrity of pancreatic tissues architecture. Integrins are cell adhesion receptors that play an integral role in cell-cell and cell-ECM communication in many cell types. β integrin associates with a subunits and mediates cellular binding to multiple extracellular matrix proteins (i.e., collagens, laminin, fibronectin). Stimulation of integrin down-stream signaling pathways are important for cell migration, differentiation, proliferation, and survival. The present study examines the currently limited knowledge regarding the contributions of integrin receptor signaling in human PaSCs function and survival.Part One:Characterization, isolation and identification of pancreatic stellate cells from human fetal pancreasObjective:To illustrate the morphology and location of PaSCs in human fetal pancreas, isolate and identify the PaSCs from human fetal pancreas.Methods:(1) Human fetal pancreatic tissues (fetal age 8-21 weeks) were collected according to protocols approved by the Health Sciences Research Ethics Board at Western University (London, ON, Canada), in accordance with the Canadian Council on Health Sciences Research Involving Human Subjects guidelines. Tissues were immediately processed for transmission electron microscopy (TEM) and immunohistology. (2) Semi-thin (500 nm) and ultra-thin (60 nm) sections were prepared, and pancreata collected from 8 to 21 weeks of fetal age were processed for TEM and morphology analysis to determine PaSC’s distribution. (3) To examine the location of PaSCs in the developing human fetal pancreas, double immunofluorescence staining of PaSCs specific markers with CK19 or insulin was performed. (4) PaSCs from human fetal pancreas (17-21 fetal age) were isolated and cultured using a modified outgrowth method. (5) The outgrown human fetal PaSCs were characterized by phase-contrast (PH) micrograph. (6) The purity of PaSCs was assessed after the 2nd passage by immunofluorescence staining and western blot analysis for stellate cell selective markers.Results:(1) The presence of PaSCs in the developing human pancreas was evidenced by TEM based on the morphology structure with foot processes located besides the ductal cells and islet cluster and verified by a double immunofluorescence staining for PaSCs marker (desmin, aSMA, GFAP, nestin, vimentin) with CK19 or insulin. (2) The PaSCs with lipid droplets surrounding a central nucleus grew out from the cell clusters over a 72 hour period, and form monolayers within 2 weeks of the culture. (3) Maturation of human fetal PaSCs was characterized by specific stellate cell selective markers (desmin, vimentin, GFAP, and aSMA), matrix proteins (collagen Ⅰ and Ⅳ, laminin, and fibronectin). These cells were also producing TGFβ1 and CTGF indicative of their intact functionalities. (4) Absence of cytokeratin 19 (a duct cell marker) and stromal cell surface marker verified the purity of isolated human PaSCs from 2 to 5 passages.Conclusions:We firstly characterized the morphology and location of PaSCs in human fetal pancreas, and demonstrated that PaSCs with foot processes located besides the ductal cells and islet cluster. Firstly isolated and identified PaSCs from fetal pancreas, indicating that purified PaSCs isolated from human fetal pancreata could be used for the following designed experiments.Part Two:Expression and function of αβ1 integrins in human fetal pancreatic stellate cellsObjective:To examine the expression of integrin receptors in the human fetal PaSCs and evaluate the effect of integrin-ECM interaction on the cell activation and proliferation.Methods:(1) β1 and β3 integrins and their associated α subunits, including al-3, a5-6, and av, were examined in isolated human PaSCs using immunofluorescence staining. (2) PaSCs were plated on 96-well tissue culture plate pre-coated with 5μg/ml of either collagen Ⅰ, collagen Ⅳ, fibronectin, laminin, or control, then adhesion and wound-healing assays were performed to evaluate cell adhesion and migration. (3) PaSCs were cultured either on collagen Ⅰ coated or non-coated plate for 24 hours and examined the proliferative capacity using immunofluorescence staining with Ki67 and growth factors production (TGFβ1 and CTGF) by western blot. (4) The expression of α3,β1 subunit integrins and involved signaling pathway were assessed by western blot.Results:(1) We found that human fetal PaSCs displayed the expression of α1-3, α5-6 and av subunits along with β1 integrin, with relatively lower β3 expression. Heterodimer α3/β1 and α5/β1 integrins are highly expressed as revealed by double immunofluorescence, which was subsequently verified by western blot. (2) Human fetal PaSCs showed a strong adhesion [(3.568±0.5677) & (2.957±0.5541) vs. (1.000 ±0.03604),p<0.01-0.001] and migration activities on collagen Ⅰ and collagen Ⅳ compare to the control. (3) Analysis of Ki67 labeling on human fetal PaSCs cultured on collagen Ⅰ showed a significantly increased proliferation capacity when compared to control [(12.44±3.028) vs. (6.303±1.702), p<0.05]. PaSCs cultured on collagen Ⅰ demonstrated elevated protein levels of TGFβ1 [(3.114±0.4693) vs. (1.000±0.1583), p<0.01] and CTGF [(1.509±0.3355) vs. (1.000±0.2980), p<0.05] relative to the control group. (4) Protein expression of β1 integrin [(2.078±0.06824) vs. (1.000± 0.3220),p<0.01] and α3 integrin [(1.336±0.2262) vs. (1.000±0.2627), p<0.05] were significantly increased in human fetal PaSCs cultured on collagen Ⅰ, and this was associated with increased phospho-FAK [(2.026±0.3769) vs. (1.000±0.08664), p<0.05] compared to the control. Furthermore, increasing activation of α3β1/FAK resulted in a significant increase of phospho-ERK1/2 [(1.542±0.6009) vs. (1.000± 0.6367),p<0.01] and phospho-AKT [(3.026±0.6649) vs. (1.000±0.3501),p<0.01], along with an increase of cyclin D1 [(1.186±0.2012) vs. (1.000±0.1933),p<0.001] protein expression compared to control group.Conclusions:Human fetal PaSCs highly express α3 and β1 integrin. Collagen Ⅰ interaction with α3β1 integrin led to enhanced PaSCs activation and proliferation via FAK/ERK and PI3K/AKT signaling pathway.Part Three:Blocking β1 integrin reduces collagen Ⅰ stimulated PaSCs function and proliferationObjective:To determine how β1 integrin is involved in regulating the function and proliferation of PaSCs via the binding of collagen Ⅰ.Methods:(1) PaSCs were pre-incubated with either anti-human β1 integrin antibody (CD29,5μg/ml), human IgG2a isotype-matched negative control (5 μg/ml), or fresh medium for 1 hour prior to seeding on collagen I coated tissue culture plates and cultured with DEMEM/Ham’s F12 plus 1% BSA medium for 24 hours. (2) At the end of the culture period, cells were subjected to adhesion and wound-healing assays in order to evaluate cell adhesion and migration. (3) For quantitative analysis of proliferative capacity in PaSCs, cells were stained for Ki67. The production (TGFβ1 and CTGF) of PaSCs was examined by western blot. (4) Involved signaling pathways were assessed by western blot.Results:(1) PaSCs treated with anti-β1 integrin displayed a 50% reduction of cell adhesion to collagen I when compared with control and IgG groups, respectively [(0.4750±0.02372) vs. (1.000±0.06726) & (0.9275±0.05621),p<0.01-0.001]. Functional blockade of β1 integrin on PaSCs severely hampered their ability to migrate and cover the gaps on collagen I matrix. No apparent difference in PaSCs adhesion [(0.9275±0.05621) vs. (1.000±0.06726),p>0.05] and migration was observed between IgG and control groups. (2) A significant decrease of Ki67+ labeling in PaSCs cultured on collagen I matrix was observed following anti-β1 integrin treatment when compared to controls [(4.913±0.4373) vs. (11.76±1.127) & (9.957±1.041), p<0.05-0.01]. Furthermore, blocking β1 integrin on PaSCs showed relatively reduced TGFβ1 [(0.3932±0.06421) vs. (1.000±0.1464) & (0.9505± 0.2482), p<0.05] and CTGF [(0.5914±0.1294) vs. (1.000±0.08527) & (1.154± 0.1290), p<0.05-0.01] protein levels when compared to the control. (3) Perturbing β1 integrin function decreased phosphorylation of FAK [(0.2320±0.03216) vs. (1.000± 0.2324) & (0.9152±0.1274), p<0.05], ERK1/2 [(0.3765±0.05115) v.s. (1.000± 0.1118) & (1.142±0.1313),p<0.05-0.01], but not AKT activation in PaSCs cultured on collagen Ⅰ [(1.218±0.1099) vs. (1.000±1.229) & (1.239±0.1276), p>0.05]. Down-regulation of FAK/ERK signaling pathways on PaSCs induced by β1 integrin blockade led to reduced cyclin D1 expression [(0.6149±0.09029) vs. (1.000± 0.08192) & (0.9749±0.04767),p<0.05-0.01].Conclusions:This β1 integrin blocking study indicates that β1 integrin interactions with the collagen Ⅰ matrix are essential for modulating human fetal PaSCs proliferation and function through FAK/ERK/cyclinDl signaling cascades. |
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