Transforming Growth Factor Beta-1 (TGF-β1) is a pleiotropic cytokine that is of central importance in wound healing, inflammation, and in key pathological processes including cancer and progressive tissue fibrosis. TGF-β1 is post-transcriptionally regulated, but the underlying mechanisms remain incompletely defined. Previously, we have extensively delineated post-transcriptional regulation of TGF-β1 synthesis in the kidney, with evidence for relief of translational repression in proximal tubular cells in the context of diabetic nephropathy. In this study, we have investigated the role of the TGF-β1 3′Untranslated Region (3′UTR). Two different 3′UTR lengths have been reported for TGF-β1, of 543 and 137 nucleotides. Absolute quantification showed that, while both UTR lengths were detectable in various human cell types and in a broad range of tissues, the short form predominated in the kidney and elsewhere. Expression of both forms was up-regulated following auto-induction by TGF-β1, but the short:long UTR ratio remained constant. Incorporation of the short UTR into a luciferase reporter vector significantly reduced reporter protein synthesis without major effect on RNA amount, suggesting post-transcriptional inhibition. In silico approaches identified multiple binding sites for miR-744 located in the proximal TGF-β1 3′UTR. A screen in RNA from human tissues showed widespread miR-744 expression. miR-744 transfection inhibited endogenous TGF-β1 synthesis, while direct targeting of TGF-β1 was shown in separate experiments, in which miR-744 decreased TGF-β1 3′UTR reporter activity. This work identifies miR-744-directed post-transcriptional regulation of TGF-β1 which, given the pleiotropic nature of cellular responses to TGF-β1, is potentially widely significant.

During the initiation and progression of fibrosis there is extensive differentiation of cells to a myofibroblastic phenotype. Because the synthesis of hyaluronan (HA) was recently linked to oncogenic epithelial-mesenchymal transformation, the present study investigated whether increased HA synthesis was also associated with myofibroblastic differentiation. HA synthesis and size were measured by incorporation of [³H]glucosamine, ion exchange, and size exclusion chromatography. Hyaluronan synthase (HAS) or hyaluronidase (HYAL) mRNA levels were assessed by reverse transcription-PCR. HYAL was detected by immunoblotting and the degradation of [³H]HA. Between 2- and 3-fold more HA appeared in the conditioned medium and became associated with the cells upon myofibroblastic differentiation. Inhibition of HAS and examination of HAS mRNA expression demonstrated that this was not the result of increased synthesis of HA or the induction of HAS 2. After differentiation, however, myofibroblasts metabolized exogenously supplied [³H]HA at a slower rate than fibroblasts and expressed lower levels of both HYAL 1 and HYAL 2 mRNA. Immunoblotting revealed more HYAL 1 and 2 in the myofibroblast conditioned medium. After acidification, however, there was no difference in HA degradation. This suggests that much of the released HYAL is inactive and that the observed differences in HA degradation are caused by cell-associated rather than secreted activity. This was confirmed by immunohistochemical staining for HYAL 1 and HYAL 2. This finding indicates the potential importance of the HYAL enzymes in controlling fibrotic progression and contrasts HA synthesis as a mediator of oncogenic transformation with that of HA degradation controlling fibrogenic differentiation.

Damage to endothelial glycocalyx impairs vascular barrier function and may contribute to progression of chronic vascular disease. An early indicator is microalbuminuria resulting from glomerular filtration barrier damage. We investigated the contributions of hyaluronic acid (HA) and chondroitin sulfate (CS) to glomerular microvascular endothelial cell (GEnC) glycocalyx and examined whether these are modified by vascular endothelial growth factors A and C (VEGFA and VEGFC). HA and CS were imaged on GEnCs and their resynthesis was examined. The effect of HA and CS on transendothelial electrical resistance (TEER) and labeled albumin flux across monolayers was assessed. Effects of VEGFA and VEGFC on production and charge characteristics of glycosaminoglycan (GAG) were examined via metabolic labeling and liquid chromatography. GAG shedding was quantified using Alcian Blue. NDST2 expression was examined using real-time PCR. GEnCs expressed HA and CS in the glycocalyx. CS contributed to the barrier to both ion (TEER) and protein flux across the monolayer; HA had only a limited effect. VEGFC promoted HA synthesis and increased the charge density of synthesized GAGs. In contrast, VEGFA induced shedding of charged GAGs. CS plays a role in restriction of macromolecular flux across GEnC monolayers, and VEGFA and VEGFC differentially regulate synthesis, charge, and shedding of GAGs in GEnCs. These observations have important implications for endothelial barrier regulation in glomerular and other microvascular beds.