Marco Pontoglio

Hepatocyte Nuclear Factor 1 Inactivation Results in Hepatic Dysfunction, Phenylketonuria, and Renal Fanconi Syndrome

HNF1 is a transcriptional activator of many hepatic genes including albumin, alpha1-antitrypsin, and alpha- and beta-fibrinogen. It is related to the homeobox gene family and is predominantly expressed in liver and kidney. Mice lacking HNF1 fail to thrive and die around weaning after a progressive wasting syndrome with a marked liver enlargement. The transcription rate of genes like albumin and alpha1-antitrypsin is reduced, while the gene coding for phenylalanine hydroxylase is totally silent, giving rise to phenylketonuria. Mutant mice also suffer from severe Fanconi syndrome caused by renal proximal tubular dysfunction. The resulting massive urinary glucose loss leads to energy and water wasting. HNF1-deficient mice may provide a model for human renal Fanconi syndrome.

Defective planar cell polarity in polycystic kidney disease

Morphogenesis involves coordinated proliferation, differentiation and spatial distribution of cells. We show that lengthening of renal tubules is associated with mitotic orientation of cells along the tubule axis, demonstrating intrinsic planar cell polarization, and we demonstrate that mitotic orientations are significantly distorted in rodent polycystic kidney models. These results suggest that oriented cell division dictates the maintenance of constant tubule diameter during tubular lengthening and that defects in this process trigger renal tubular enlargement and cyst formation.

Defective insulin secretion in hepatocyte nuclear factor 1alpha-deficient mice.

Mutations in the gene for the transcription factor hepatocyte nuclear factor (HNF) 1alpha cause maturity-onset diabetes of the young (MODY) 3, a form of diabetes that results from defects in insulin secretion. Since the nature of these defects has not been defined, we compared insulin secretory function in heterozygous [HNF-1alpha (+/-)] or homozygous [HNF-1alpha (-/-)] mice with null mutations in the HNF-1alpha gene with their wild-type littermates [HNF-1alpha (+/+)]. Blood glucose concentrations were similar in HNF-1alpha (+/+) and (+/-) mice (7.8+/-0.2 and 7.9+/-0.3 mM), but were significantly higher in the HNF-1alpha (-/-) mice (13.1+/-0.7 mM, P < 0.001). Insulin secretory responses to glucose and arginine in the perfused pancreas and perifused islets from HNF-1alpha (-/-) mice were < 15% of the values in the other two groups and were associated with similar reductions in intracellular Ca2+ responses. These defects were not due to a decrease in glucokinase or insulin gene transcription. beta cell mass adjusted for body weight was not reduced in the (-/-) animals, although pancreatic insulin content adjusted for pancreas weight was slightly lower (0.06+/-0.01 vs. 0.10+/-0.01 microg/mg, P < 0.01) than in the (+/+) animals. In summary, a null mutation in the HNF-1alpha gene in homozygous mice leads to diabetes due to alterations in the pathways that regulate beta cell responses to secretagogues including glucose and arginine. These results provide further evidence in support of a key role for HNF-1alpha in the maintenance of normal beta cell function.

A transcriptional network in polycystic kidney disease

Mutations in cystic kidney disease genes represent a major genetic cause of end‐stage renal disease. However, the molecular cascades controlling the expression of these genes are still poorly understood. Hepatocyte Nuclear Factor 1β (HNF1β) is a homeoprotein predominantly expressed in renal, pancreatic and hepatic epithelia. We report here that mice with renal‐specific inactivation of HNF1β develop polycystic kidney disease. We show that renal cyst formation is accompanied by a drastic defect in the transcriptional activation of Umod, Pkhd1 and Pkd2 genes, whose mutations are responsible for distinct cystic kidney syndromes. In vivo chromatin immunoprecipitation experiments demonstrated that HNF1β binds to several DNA elements in murine Umod, Pkhd1, Pkd2 and Tg737/Polaris genomic sequences. Our results uncover a direct transcriptional hierarchy between HNF1β and cystic disease genes. Interestingly, most of the identified HNF1β target gene products colocalize to the primary cilium, a crucial organelle that plays an important role in controlling the proliferation of tubular cells. This may explain the increased proliferation of cystic cells in MODY5 patients carrying autosomal dominant mutations in HNF1β.

Hepatic Stem-like Phenotype and Interplay of Wnt/β-Catenin and Myc Signaling in Aggressive Childhood Liver Cancer

Hepatoblastoma, the most common pediatric liver cancer, is tightly linked to excessive Wnt/beta-catenin signaling. Here, we used microarray analysis to identify two tumor subclasses resembling distinct phases of liver development and a discriminating 16-gene signature. beta-catenin activated different transcriptional programs in the two tumor types, with distinctive expression of hepatic stem/progenitor markers in immature tumors. This highly proliferating subclass was typified by gains of chromosomes 8q and 2p and upregulated Myc signaling. Myc-induced hepatoblastoma-like tumors in mice strikingly resembled the human immature subtype, and Myc downregulation in hepatoblastoma cells impaired tumorigenesis in vivo. Remarkably, the 16-gene signature discriminated invasive and metastatic hepatoblastomas and predicted prognosis with high accuracy.

HNF1α controls renal glucose reabsorption in mouse and man

Recently it has been shown that dominant mutations in the human hepatocyte nuclear factor 1 α (HNF1α) gene, encoding for a homeoprotein that is expressed in liver, kidney, pancreas and intestine, result in maturity onset diabetes of the young type 3 (MODY3). HNF1α‐null mice are diabetic, but at the same time suffer from a renal Fanconi syndrome characterized by urinary glucose loss. Here we show that MODY3 patients are also characterized by a reduced tubular reabsorption of glucose. The renal murine defect is due to reduced expression of the low affinity/high capacity glucose cotransporter (SGLT2). Our results show that HNF1α directly controls SGLT2 gene expression. Together these data indicate that HNF1α plays a key role in glucose homeostasis in mammals.

Transcription factor-dependent regulation of CBP and P/CAF histone acetyltransferase activity.

CREB‐binding protein (CBP) and CBP‐associated factor (P/CAF) are coactivators possessing an intrinsic histone acetyltransferase (HAT) activity. They are positioned at promoter regions via association with sequence‐specific DNA‐binding factors and stimulate transcription in a gene‐specific manner. The current view suggests that coactivator function depends mainly on the strength and specificity of transcription factor–coactivator interactions. Here we show that two dominant‐negative mutants of hepatocyte nuclear factor‐1α (HNF‐1α), P447L and P519L, occurring in maturity onset diabetes of the young (MODY3) patients, exhibit paradoxically stronger interactions than the wild‐type protein with either CBP or P/CAF. However, CBP and P/CAF recruited by these mutants lack HAT activity. In contrast, wild‐type HNF‐1α and other transcription factors, such as Sp1 or HNF‐4, stimulated the HAT activity of CBP. The results suggest a more dynamic role for DNA‐binding proteins in the transcription process than was considered previously. They are not only required for the recruitment of coactivators to the promoter but they may also modulate their enzymatic activity.

AKT2 is essential to maintain podocyte viability and function during chronic kidney disease

In chronic kidney disease (CKD), loss of functional nephrons results in metabolic and mechanical stress in the remaining ones, resulting in further nephron loss. Here we show that Akt2 activation has an essential role in podocyte protection after nephron reduction. Glomerulosclerosis and albuminuria were substantially worsened in Akt2−/− but not in Akt1−/− mice as compared to wild-type mice. Specific deletion of Akt2 or its regulator Rictor in podocytes revealed that Akt2 has an intrinsic function in podocytes. Mechanistically, Akt2 triggers a compensatory program that involves mouse double minute 2 homolog (Mdm2), glycogen synthase kinase 3 (Gsk3) and Rac1. The defective activation of this pathway after nephron reduction leads to apoptosis and foot process effacement of the podocytes. We further show that AKT2 activation by mammalian target of rapamycin complex 2 (mTORC2) is also required for podocyte survival in human CKD. More notably, we elucidate the events underlying the adverse renal effect of sirolimus and provide a criterion for the rational use of this drug. Thus, our results disclose a new function of Akt2 and identify a potential therapeutic target for preserving glomerular function in CKD.

A mitotic transcriptional switch in polycystic kidney disease

Hepatocyte nuclear factor-1β (HNF-1β) is a transcription factor required for the expression of several renal cystic genes and whose prenatal deletion leads to polycystic kidney disease (PKD). We show here that inactivation of Hnf1b from postnatal day 10 onward does not elicit cystic dilations in tubules after their proliferative morphogenetic elongation is over. Cystogenic resistance is intrinsically linked to the quiescent state of cells. In fact, when Hnf1b deficient quiescent cells are forced to proliferate by an ischemia-reperfusion injury, they give rise to cysts, owing to loss of oriented cell division. Remarkably, in quiescent cells, the transcription of crucial cystogenic target genes is maintained even in the absence of HNF-1β. However, their expression is lost as soon as cells proliferate and the chromatin of target genes acquires heterochromatin marks. These results unveil a previously undescribed aspect of gene regulation. It is well established that transcription is shut off during the mitotic condensation of chromatin. We propose that transcription factors such as HNF-1β might be involved in reprogramming gene expression after transcriptional silencing is induced by mitotic chromatin condensation. Notably, HNF-1β remains associated with the mitotically condensed chromosomal barrels. This association suggests that HNF-1β is a bookmarking factor that is necessary for reopening the chromatin of target genes after mitotic silencing.

Anatomy of a Homeoprotein Revealed by the Analysis of Human MODY3 Mutations

Hepatocyte nuclear factor 1α (HNF1α) is an atypical dimeric homeodomain-containing protein that is expressed in liver, intestine, stomach, kidney, and pancreas. Mutations in the HNF1α gene are associated with an autosomal dominant form of non-insulin-dependent diabetes mellitus called maturity-onset diabetes of the young (MODY3). More than 80 different mutations have been identified so far, many of which involve highly conserved amino acid residues among vertebrate HNF1α. In the present work, we investigated the molecular mechanisms by which MODY3 mutations could affect HNF1α function. For this purpose, we analyzed the properties of 10 mutants resulting in amino acid substitutions or protein truncation. Some mutants have a reduced protein stability, whereas others are either defective in the DNA binding or impaired in their intrinsic trans-activation potential. Three mutants, characterized by a complete loss of trans-activation, behave as dominant negatives when transfected with the wild-type protein. These data define a clear causative relationship between MODY3 mutations and functional defects in HNF1α trans-activation. In addition, our analysis sheds new light on the structure of a homeoprotein playing a key role in pancreatic β cell function.