Study of the two UDPglucose::glycoprotein glucosyltransferases homologues in the maintenance of the intestinal homeostasis and in the mammary gland development in the mouse

ACOSTA-MONTALVO G; RIAL HAWILA MR; BOTTERO E; CASTRO O.; MIRANDA S

CABA

Congreso; 2do Congreso Internacional en Medicina Traslacional. Cellular and Molecular pathways as therapeutic targets; 2015

FFYB.UBA

UGGT (UDP-Glc::glycoproteinglucosyltransferase) is the key component of the quality control mechanism ofglycoprotein folding that ensures that only properly folded proteins exit theER. The accumulation of misfolded proteins in the ER, state known as ¡°ERstress¡±, triggers a complex response called UPR (¡°unfolded protein response¡±)that drives the cell to homeostasis in normal conditions or, in case ofpersistent ER stress, leads it to dysfunction, tissue damage and chronicinflammatory diseases such as in IBD (¡°inflammatory bowel disease¡±).  We have previously described a second isoform in mouse and C.elegans. Both of them show canonical UGGT activity during in vitro assays, but they likely play different biologicalfunctions. To analyze this hypothesis, we investigated their expression in amouse model of intestinal inflammation and in mammary gland development. Tothis aim, antiserum anti-UGGT2 was raised in rabbits employing a multiantigenicpeptide synthesized with a sequence of UGGT2 (NP_001074721.2) not present inUGGT1 (NP_942602.2). The first model consists of exposing mice to 24h of anaccoustic exposure -AS- (300Hz-70dB). Mice were killed: 1) after AS; 2) threeweeks after AS; 3) mice were submitted to AS once a week during a month andthen were killed. Control groups did not receive AS. Sections from smallintestine were employed for stainings (H&E, May Gr¨¹nwald-Giemsa andPAS/Hematoxylin), immunohistochemical (for detecting TNF¦Á,  TGF-¦Â ad Ki67) and immunofluorescence studies(for CCL25, IL-17 and IL-22). UGGT1/UGGT2 expression was analyzed byimmunohistochemistry. Results showed that AS induced inflammation and several histologicalalterations. UGGTs are moderately expressed in normal conditions. After one (1)or various AS (3), UGGT1/UGGT2 expression increased proportionally toinflammation (for both: group 3>1, p<0.001), even though the cytokineexpression pattern was different between these groups. Three weeks after AS(2), the histological alterations exacerbated, UGGT1 maintained its highexpression while UGGT2 was downregulated (group 2<1, p<0.01; group 2<3,p<0.001). Taking into account that previous results showed thatprogesterone regulates the expression and activity of UGGTs isoforms, we investigatedtheir expression in mammary gland development during pregnancy in comparison tovirgin C57BL/6 females and in cultures of T47D cells in the presence ofprogesterone (0, 10-5, 10-6, 10-7 and 10-8M).In both models, we determined the expression of uggt1 and uggt2 mRNA by qRTPCR.In addition, the expression of both UGGTs was analyzed by immunohistochemistry.Results showed that the expression of uggt1 and uggt2 in T47D cells peaked athigh levels of progesterone. Moreover, UGGT1 is expressed by mammary epithelialcells while UGGT2 is expressed by mioepithelial cells.   Conclusions: UGGTs may play a physiologic role in the gut andin the mammary gland. The gut model showed that they would respond to differentsignals and/or have distinct half-life while our studies in mammary cells aredifferentially regulated by progesterone.