SUMMARYThe mammalian kidney is composed of thousands of individual epithelial tubules known as nephrons. Deficits in nephron number are associated with myriad diseases ranging from complete organ failure to congenital hypertension. A balance between differentiation and maintenance of a mesenchymal progenitor cell population determines the final number of nephrons. How this balance is struck is poorly understood. Previous studies have suggested that Wnt9b/-catenin signaling induced differentiation (mesenchymal-to-epithelial transition) in a subset of the progenitors but needed to be repressed in the remaining progenitors to keep them in the undifferentiated state. Here, we report that Wnt9b/-catenin signaling is active in the progenitors and is required for their renewal/proliferation. Using a combination of approaches, we have revealed a mechanism through which cells receiving the same Wnt9b/-catenin signal can respond in distinct ways (proliferate versus differentiate) depending on the cellular environment in which the signal is received. Interpretation of the signal is dependent, at least in part, on the activity of the transcription factor Six2. Six2-positive cells that receive the Wnt9b signal are maintained as progenitors whereas cells with reduced levels of Six2 are induced to differentiate by Wnt9b. Using this simple mechanism, the kidney is able to balance progenitor cell expansion and differentiation insuring proper nephron endowment. These findings provide novel insights into the molecular mechanisms that regulate progenitor cell differentiation during normal and pathological conditions.

Transcriptional activation of eukaryotic genes depends on the precise and ordered recruitment of activators, chromatin modifiers/remodelers, coactivators, and general transcription factors to the promoters of target genes. Using the human matrix metalloproteinase 9 (MMP-9) gene as a model system, we investigated the sequential assembly and dynamic formation of transcription complexes on a human promoter under the influence of mitogen signaling. We find that, coincident with activation of the MMP-9 gene, activators, chromatin remodeling complexes, and coactivators are recruited to the preassembled MMP-9 promoter in a stepwise and coordinated order, which is dependent on activation of MEK-1/extracellular signal-regulated kinase and NF-B signaling pathways. Conversely, corepressor complexes are released from the MMP-9 promoter after transcriptional activation. Histone modifications shift from repressive to permissive modifications concurrent with activation of the MMP-9 gene. Chromatin remodeling induced by Brg-1 is required for MMP-9 gene transcription, which is concomitant with initiation of transcription. Therefore, coordination of cell signaling, chromatin remodeling, histone modifications, and stepwise recruitment of transcription regulators is critical to precisely regulate MMP-9 gene transcription in a temporally and spatially dependent manner. Given the important role of MMP-9 in both normal development and pathological conditions, understanding MMP-9 gene regulation is of great relevance.Gene transcription in eukaryotic cells is controlled by protein complexes, including general and tissue-specific transcription factors, coregulators, chromatin-remodeling complexes, and complexes responsible for signal-specific histone modifications (26). As eukaryotic DNA is packaged into chromatin, generally a repressive structure for transcriptional activation, transcription in the context of chromatin requires remodeling processes to reconfigure the chromatin, so that activators, coactivators, and general transcription factors (GTFs) have access to promoters of target genes (12). Chromatin remodeling is dependent on either ATP-dependent chromatin-remodelingcomplex-induced structural modifications of nucleosomes or histone acetyltransferase-(HAT) and histone methyltransferase-mediated covalent modifications of the N-terminal tails of core histones (12). The SWI/SNF chromatin-remodeling complex can alter chromatin structure by either shifting nucleosomes along the DNA or twisting DNA to modulate the nucleosome structure (42). Brg-1 and Brm are two ATPase subunits of the SWI/SNF complex. Recruitment of the SWI/ SNF complex to target promoters requires protein-protein interactions through Brg-1 and other transcription regulators, as Brg-1 does not recognize sequence-specific DNA (21).

Polycystic kidney disease (PKD) is a genetic disorder that is characterized by cyst formation in kidney tubules. PKD arises from abnormalities of the primary cilium, a sensory organelle located on the cell surface. Here, we show that the primary cilium of renal epithelial cells contains a protein complex comprising adenylyl cyclase 5/6 (AC5/6), A-kinase anchoring protein 150 (AKAP150), and protein kinase A. Loss of primary cilia caused by deletion of Kif3a results in activation of AC5 and increased cAMP levels. Polycystin-2 (PC2), a ciliary calcium channel that is mutated in human PKD, interacts with AC5/6 through its C terminus. Deletion of PC2 increases cAMP levels, which can be corrected by reexpression of wild-type PC2 but not by a mutant lacking calcium channel activity. Phosphodiesterase 4C (PDE4C), which catabolizes cAMP, is also located in renal primary cilia and interacts with the AKAP150 complex. Expression of PDE4C is regulated by the transcription factor hepatocyte nuclear factor-1β (HNF-1β), mutations of which produce kidney cysts. PDE4C is down-regulated and cAMP levels are increased in HNF-1β mutant kidney cells and mice. Collectively, these findings identify PC2 and PDE4C as unique components of an AKAP complex in primary cilia and reveal a common mechanism for dysregulation of cAMP signaling in cystic kidney diseases arising from different gene mutations.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play crucial roles in proteolytic degradation of the extracellular matrix. Aberrant expression of the 92-kDa type IV collagenase (MMP-9) is implicated in the invasion and angiogenesis process of malignant tumors and in inflammatory diseases of the CNS. We investigated the effects of IFN-γ and IFN-β, cytokines used for treating some cancers and multiple sclerosis, on MMP-9 expression in human astroglioma and fibrosarcoma cell lines and primary astrocytes. Our results demonstrate that IFN-γ and IFN-β significantly inhibit MMP-9 enzymatic activity and protein expression that is induced by PMA and the cytokine TNF-α. The inhibitory effects of IFN-γ and IFN-β on MMP-9 expression correlate with decreased steady state MMP-9 mRNA levels and suppression of MMP-9 promoter activity. IFN-γ- and IFN-β-mediated inhibition of MMP-9 gene expression is dependent on the transcription factor STAT-1α, since IFN-γ and IFN-β fail to suppress MMP-9 expression in STAT-1α-deficient primary astrocytes and human fibrosarcoma cells. Reconstitution of human STAT-1α successfully restores the inhibitory effects of IFN-γ and IFN-β on MMP-9 gene expression. Thus, these data demonstrate the critical role of STAT-1α in IFN-γ and IFN-β suppression of MMP-9 gene expression.

Hepatocyte nuclear factor-1␤ (HNF-1␤) is a Pit-1, Oct-1/2, Unc-86 (POU) homeodomain-containing transcription factor expressed in the kidney, liver, pancreas, and other epithelial organs. Mutations of HNF-1␤ cause maturity-onset diabetes of the young, type 5 (MODY5), which is characterized by early-onset diabetes mellitus and congenital malformations of the kidney, pancreas, and genital tract. Knockout of HNF-1␤ in the mouse kidney results in cyst formation. However, the signaling pathways and transcriptional programs controlled by HNF-1␤ are poorly understood. Using genome-wide chromatin immunoprecipitation and DNA microarray (ChIP-chip) and microarray analysis of mRNA expression, we identified SOCS3 (suppressor of cytokine signaling-3) as a previously unrecognized target gene of HNF-1␤ in the kidney. HNF-1␤ binds to the SOCS3 promoter and represses SOCS3 transcription.