Wnt-4 is a secreted glycoprotein that is critical for genitourinary development but found only in the most distal collecting duct epithelium in the normal murine adult kidney. Wnt4 expression is induced throughout the collecting ducts in four murine models of renal injury that produce tubulointerstitial fibrosis: folic acid-induced nephropathy, unilateral ureteral obstruction, renal needle puncture, and genetic polycystic kidney disease. Wnt4 activation induced by injury is limited to collecting ducts, with initial activation in the collecting duct epithelium followed by activation in fibrotic lesions surrounding the collecting ducts. The highest cellular Wnt4 expression is in interstitial fibroblasts in the fibrotic lesions that also express high levels of collagen-α1(I) mRNA and α-smooth muscle actin. In support of a functional role for Wnt-4 in these activated myofibroblasts, Wnt-4 induces stabilization of cytosolic β-catenin in a cultured myofibroblast cell line. Furthermore, Wnt-4-producing fibroblasts placed under the renal capsule of adult mice induce lesions with tubular epithelial destruction. These observations suggest a role for Wnt-4 in the pathogenesis of renal fibrosis.

Hepatocyte nuclear factor (HNF)-1alpha plays a central role in intestinal and hepatic gene regulation and is required for hepatic expression of the liver fatty acid binding protein gene (Fabpl). An Fabpl transgene was directly activated through cognate sites by HNF-1alpha and HNF-1beta, as well as five other endodermal factors: CDX-1, C/EBPbeta, GATA-4, FoxA2, and HNF-4alpha. HNF-1alpha activated the Fabpl transgene by as much as 60-fold greater in the presence of the other five endodermal factors than in their absence, accounting for up to one-half the total transgene activation by the group of six factors. This degree of synergistic interaction suggests that multifactor cooperativity is a critical determinant of endodermal gene activation by HNF-1alpha. Mutations in HNF-1alpha that result in maturity onset diabetes of the young (MODY3) provide evidence for the in vivo significance of these synergistic interactions. An R131Q HNF-1alpha MODY3 mutant exhibits complete loss of synergistic activation in concert with the other endodermal transcription factors despite wild-type transactivation ability in their absence. Furthermore, whereas wild-type HNF-1alpha exhibited pairwise cooperative synergy with each of the other five factors, the R131Q mutant could synergize only with GATA-4 and C/EBPbeta. Selective loss of synergy with other endodermal transcription factors accompanied by retention of native transactivation ability in an HNF-1alpha MODY mutant suggests in vivo significance for cooperative synergy.

Hepatic nuclear factor (HNF)-4alpha and HNF-1alpha are key endodermal transcriptional regulators that physically and functionally interact. HNF-4alpha and HNF-1alpha cooperatively activate genes with binding sites for both factors, whereas suppressive interactions occur at regulatory sequences with a binding site for only one factor. The liver fatty acid binding protein gene (Fabp1) has binding sites for both factors, and chromatin precipitation assays were utilized to demonstrate that HNF-4alpha increased HNF-1alpha Fabp1 promoter occupancy during cooperative transcriptional activation. The HNF4 P2 promoter contains a HNF-1 but not HNF-4 binding site, and HNF-4alpha suppressed HNF-1alpha HNF4 P2 activation and decreased promoter HNF-1alpha occupancy. The apolipoprotein C III (APOC3) promoter contains a HNF-4 but not HNF-1 binding site, and HNF-1alpha suppressed HNF-4alpha APOC3 activation and decreased HNF-4alpha promoter occupancy. Maturity onset diabetes of the young (MODY) as well as defects in hepatic lipid metabolism result from mutations in either HNF-4alpha or HNF-1alpha. We found that MODY missense mutant R127W HNF-4alpha retained wild-type individual Fabp1 activation and bound to HNF-1alpha better than wild-type HNF-4alpha, yet did not cooperate with HNF-1alpha or increase HNF-1alpha Fabp1 promoter occupancy. The R127W mutant was also defective in both suppressing HNF-1alpha activation of HNF4 P2 and decreasing HNF-1alpha promoter occupancy. The HNF-1alpha R131Q MODY mutant also retained wild-type Fabp1 activation and bound to HNF-4alpha as well as the wild type but was defective in both suppressing HNF-4alpha APOC3 activation and decreasing HNF-4alpha promoter occupancy. These results suggest HNF-1alpha-HNF-4alpha functional interactions are accomplished by regulating factor promoter occupancy and that defective factor-factor interactions may contribute to the MODY phenotype.

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