Stromal-derived HGF Supports The Proliferation Of Lung Progenitor Cells Through Repression Of Smad4

Objective:Using the present techniques,we knocked out Smad4 in lung epithelial progenitor cells.We adopted an in vitro epithelial-fibroblast co-culture assaydeveloped previously to observe that the TGF?-HGF-Smad axis regulates the proliferation of lung progenitor cells.Method:Our previous study has proved that inhibition of the TGF?signal pathway by SB43154 activates the production of growth factor FGFs in fibroblasts.FGFs can promote the proliferation of lung epithelium progenitor cells.Firstly,fibroblasts have been shown to produce growth factors that could promote lung epithelial progenitor cells.In order to study whether HGF is regulated by TGF?,we cultured MLg cells with or without SB431542.Using quantitative RT-PCR,we observed the expression of messenger RNAs for HGF in the SB431542 treatment.Secondly,we sorted out the mouse lung progenitor cells,which total RNA were extracted,then we preformed qPCR to detect the RNA expression level of HGF receptor,c-Met.Thirdly,EpCAM⁺lung epithelial cells or EpCAM⁺Sca-1⁺lung progenitor cells were sorted and pooled for total RNA extraction,Data were annotated with Affymetrix Expression Console software?Affymetrix?.Lastly,Sftpc-Cre mice and Smad4^f/f/f mice were crossed to generate Sftpc-Cre;Smad4^f/f/f mice.Lung progenitor cells from Sftpc-Cre^-;Smad4^f/f or Sftpc-Cre⁺;Smad4^f/f mice were sorted and cultured in the presence of MLg cells.We observed epithelial colonies in the Sftpc-Cre⁺;Smad4^f/f group,and compared to the Sftpc-Cre^-;Smad4^f/f control group in the absence of SB431542.Results:?1?We then measured HGF expression in absence or presence of SB431542.Within 48 h,the number of MLg cells increased by about tenfold in the control group.There was no difference in the number of MLg cells between control and SB431542treatment.Additionally,the morphology of MLg cells did not differ between the control and SB431542 treatment.Using quantitative RT-PCR,we observed that the messenger RNA expression of Hgf in the SB431542 treatment was higher than that in the control.?2?We observed that c-Met expression is more abundant in lung epithelial cells than in stromal cells.In vitro 3-D matrigel culture indicated that HGF promotes the growth of lung progenitor cells in absence of MLg cells?3?TGF?transcript is enriched by 3.5 fold over total lung epithelial cells,Gather analysis of top genes in lung progenitor cells indicated that Smad4 signaling pathway may participate in the regulation of lung progenitor cells proliferation.?4?We observed more epithelial colonies in the Sftpc-Cre⁺;Smad4^f/f group as compared to the Sftpc-Cre^-;Smad4^f/f control group in the absence of SB431542.This change was absent,however,between these two groups in the presence of SB431542Conclusion:?1?Activation of the TGF?signal pathway inhibits the production of growth factor HGF in fibroblasts.?2?Met expression is more abundant in lung epithelial cells than in stromal cells HGF promotes the growth of lung progenitor cells in absence of MLg cells,suggesting that HGF acts directly on lung progenitor cells to promote their growth.?3?Smad4 signaling pathway may participate in the regulation of lung progenitor cell proliferation.These data suggested that Smad4 may be a regulator of lung progenitor cells.?4?TGF?activation is required for the regulation of lung progenitor cells by Smad4.We proposed that the TGF?/TGF?R1/2 axis plays an inhibitory role in the expression and/or secretion of growth factors HGF in fibroblasts.These data suggested a role for the TGF?-TGF?R1/2-HGF-Smad4 axis in lung epithelial homeostasis.In conclusion,our study suggests that SB431542 promotes the proliferation of lung progenitor cells via the TGF?receptor on fibroblasts,thereby altering their secretory properties including the secretion of HGF.Additionally,Smad4 may play a mechanistic role in the regulation of lung progenitor cells by HGF.