The Role Of The Superfamily Of TGF-β Receptor Glycosylation For The Development Of Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is associated with pronounced morbidity andmortality and responds poorly to known therapeutic interventions; there are no drugsthat effectively block or reverse progressive fibrosis. Until now there is some evidenceindicated that transforming growth factor beta (TGF-β) signaling is important foronset and development of pulmonary fibrosis in human and model animals. TGF-betasignal through transmembrane receptor serine/threonine kinase activate the novel signalintermediates called Smad proteins. And most of the pro-fibrotic activities of TGF-βareregulated by Smad2and Smad3.,ALK4,5and7(which are type I receptors of TGF-β)are similar in structure and function and would make Smad phosphonation. But nobodywould make it clear that which kind of type I receptor ALK4,5and7play a roleduring the pulmonary fibrosis development. In this study, we investigated the role ofTGF-beta type I receptors ALK4,5and7in the pathogenesis of pulmonary fibrosis inbleomycin (BLM)-induced pulmonary fibrosis model in Sprague-Dawley rats. Here, weconduct a comprehensive analysis of the function of the three type I receptors. Wefound that ALK5and TGF-βRII were glycoprotein, containing core fucose, whichsuggested the functions of TGF-β receptor were regulated by core fucose glycosylationmodification. We engineered an lung fibroblast cell line(WI-38) to examine core fucoseglycosylation, and its constitutive contributions of anti-RIF to block TGF-β/Smad2/3signal pathway. Then we found the prescription of HuoXueHuaYu chinese herbs couldaffect glycosylation of the receptor of TGF-β and the signaling pathway ofTGF-β/-Smads.Part I The Expression of ALK4,ALK5and ALK7ulmonary Fibrosis nduced by Bleomycin.Objective： To identify the distribution and expression of the subsets of TGF-βRⅠ (ALK4,ALK5and ALK7) in normal rat lung and pulmonary fibrosisinduced by bleomycin.Method：7adult pathogen-free female rats was as control group.49adultpathogen-free female SD rats were treated with bleomycin by intranasal instillation.Every seven rats were killed on the1,3,7,14,28and35days. The distribution andexpression of ALK4, ALK5and ALK7in normal lung and pulmonary fibrosis wereconfirmed by Real Time-PCR, western-blotting and Immunohistochemistry.Result： Pulmonary fibrosis was observed in the rats treated with bleomycin byhistology. The mRNA of ALK4, ALK5and ALK7showed at the first day in the ratstreated with bleomycin and elevated progressly in time-dependent manner.: Comparedwith normal group, the levels of ALK4、ALK5and ALK7were significantly increasedin the rat model of pulmonary fibrosis.ALK4is the2.5fold in14days,ALK5is the5fold in28days and ALK7is the2fold in28days.Conclusion： The expression of ALK4、ALK5and ALK7were significantlyincreased in the rat model of pulmonary fibrosis.Part II The Role of α1,6-fucosyltransferase8in TGF-β/Smad2/3Signaling Pathway in Pulmonary Fribroblast CellsObjective: To investigate the effect of FUT8siRNA on TGF-β/Smad2/3signalingpathway in pulmonary fribroblast cells.Method: WI-38cells were randomly divided into six groups:(1) normalgroup;(2)FUT8siRNA group: WI-38cells were incubated with FUT8siRNA for72h;(3)TGF group: WI-38cells were incubated with10ng/mL TGF-β1for48hs;(4)TGFM group: WI-38cells were incubated with Mock siRNA for72hs;(5)TGFF group:WI-38cells were incubated with FUT8siRNA for24hs,then with10ng/mL TGF-β1foranother48hs;(6)TGFFsF group: WI-38cells were incubated with FUT8siRNA for24hs,with10ng/mL TGF-β1for another48h, then with5ng/mL FUT824hs. The FUT8genewas silenced RNAi technology,the expression of core fucose in the WI-38cells wasdetected by immunofluorescent analysis, the core fucosylation of TGF-βRII and ALK5was detected by lectin blotting, the expression of p-Smad2/3, MMP-9and TIMP-1inWI-38cells was detected by Real-time PCR and western blotting. The expression ofα-SMA was detected by Flow cytometry. Results: Compared with the control group, incubation with5ng/mL TGF-β1for48hs could significantly upregulate the core fucosylation of WI-38cells and enhancethe expression of TGF-βRII and ALK5(P<0.05), furthermore, it could markedlyincrease the expression of p-Smad2/3(P<0.05) and MMP-9and TIMP-1, whileFUT8siRNA could inhibit the upregulation of TGF-βRII and ALK5(P<0.05), andsuppress the p-Smad2/3,α-SMA,MMP-9and TIMP-1(P<0.05) without disturbing theexpression of TGF-βRII and ALK5. This inhibition can be relieved by exogenousFUT8.Conclusion: FUT8-catalized core fucosylation of TGF-βRII and ALK5is essentialto fulfill their functions and block core fucosylation of TGF-βRII and ALK5caused theinactivation of TGF-β/Smad2/3signaling pathway in WI-38cells.Part III To Explore the Effect of the Prescription of HuoXueHuaYu Chinese herbson Core Fucosylation of TGF-β Receptor in WI-38cellsObjective: To find whether the prescription of HuoXueHuaYu chinese herbsaffect glycosylation of the receptor of TGF-β and the signaling pathway ofTGF-β/-Smad2/3.Method: The WI-38cells were divided randomly into3groups:(1)normal group:cultured in DMEM medium;(2) TGF group:cultured in10ng/ml TGF-β1medium for48hours;(3) TGFH group:cultured in10%prescription of HuoXueHuaYu chineseherbs with rat serum for24hours, then added10ng/ml TGF-β1into the medium andcultured for another48hours. The expression of FUT8, LCA, ALK5and TGF-βRⅡ inthe three groups were detected by real-time PCR, immunofluorescence and westernblotting, and the level of collagenⅠ、Ⅲ、Ⅳ，COLⅠ、Ⅲ、Ⅳ and FN of the three groupswere detected with ELISA.Result: The prescription of HuoXueHuaYu chinese herbs can reduce theexpression of FUT8and LCA in WI-38cell. Prescription of HuoXueHuaYu chineseherbs can not affect the expression of ALK5and TGF-βRⅡ but it reduced theexpression of TGF-β1on p-Smad2/3of WI-38cell. The COL Ⅰwas1.5times higher inTGF group than in normal group(p<0.05). The COLⅢ was2.5times higher in TGFgroup than in control group(p<0.05). The COLⅣ was1times higher in TGF group thanin control group(p<0.05). The FN was2.5times higher in TGF group than in control group(p<0.05). And the COL Ⅰ,COLⅢ, COLⅣ and FN in TGFH group is a littlehigher than in control group(p>0.05).Conclusion:1. The prescription of HuoXueHuaYu chinese herbs could inhibit the glycosylationthe receptor of TGF-β(ALK5and TGF-βRⅡ) and signaling pathway ofALK5/TGF-βRⅡ-smad2/3.2. The prescription of HuoXueHuaYu chinese herbs could reduce the expression ofcollegen of Ⅰ、Ⅲ、Ⅳ and FN.