Steroidal regulation of connective tissue growth factor (CCN2; CTGF) synthesis in the mouse uterus

Aims-To determine mechanisms regulating the production of connective tissue growth factor (CCN2; CTGF) and transforming growth factor beta1 (TGF-beta1 ) in the mouse uterus. Methods-In situ hybridisation and immunohistochemistry were used to localis e CCN2 (CTGF) and TGF-beta1 in uteri from sexually mature female mice that had either been (1) mated with sterile males to induce pseudopregnancy or ( 2) ovariectomised (OVX) and administered estradiol-17 beta (E-2) or progest erone (P-4), either alone or in combination. Uteri collected on days 0.5, 1 .5, 2.5, 3.5, 4.5, or 5.5 of pseudopregnancy or at one, three, six, 12, or 24 hours after steroid administration were fixed, sectioned, and incubated with specific riboprobes or antibodies to permit detection and localisation of mRNA or protein for CTGF and TGF-beta1. Results-On days 0.5-2.5 of pseudopregnancy, CCN2 (CTGF) and TGF-beta1 were principally colocalised to uterine epithelial cells, with much smaller amou nts in the stroma. On days 3.5-4.5, there was a reduction of CCN2 (CTGF) an d TGF-beta1 in the epithelium but an increase in stromal and endothelial ce lls, corresponding to a period of extracellular matrix remodelling and neov ascularisation within the endometrium. In OVX mice, epithelial cells were w eakly positive for both CCN2 (CTGF) and TGF-beta1 in the absence of steroid hormones. Epithelial CTGF mRNA production were strongly but transiently st imulated in OVX mice cells by E-2. These effects were antagonised by P-4, w hich itself transiently stimulated epithelial CCN2 (CTGF) production, altho ugh less robustly than E-2. CTGF and TGF-beta1 protein amounts were high in epithelial cells throughout steroid treatment and were increased in the st roma, where they were relatively long lived. Stromal CCN2 (CTGF) and TGF-be ta1 were lower after co-administration of E-2 and P-4 than in response to e ach hormone individually. Although ccn2 (ctgf) is a TGF-beta1 inducible gen e in other systems, and both growth factors were often co-localised in uter ine tissues in these studies, several treatment regimens resulted in high a mounts of TGF-beta1 protein in stromal cells without the concomitant produc tion of ccn2 (ctgf) mRNA. Conclusions-Maternal factors are principal cues for CCN2 (CTGF) and TGF-bet a1 production in the uterus because (1) their expression during pseudopregn ancy is comparable to that seen in pregnancy and (2) they are regulated by ovarian steroids. TGF-beta dependent and independent mechanisms of ccn2 (ct gf) gene transcription exist in the uterus that are variably regulated by s teroid hormones. Collectively, the data support a role for CCN2 (CTGF) in m ediating the effects of steroid hormones and TGF-beta on endometrial functi on.