The CDK4-pRB-E2F1 pathway controls insulin secretion.

CDK4-pRB-E2F1 cell-cycle regulators are robustly expressed in non-proliferating beta cells, suggesting that besides the control of beta-cell number the CDK4-pRB-E2F1 pathway has a role in beta-cell function. We show here that E2F1 directly regulates expression of Kir6.2, which is a key component of the K(ATP) channel involved in the regulation of glucose-induced insulin secretion. We demonstrate, through chromatin immunoprecipitation analysis from tissues, that Kir6.2 expression is regulated at the promoter level by the CDK4-pRB-E2F1 pathway. Consistently, inhibition of CDK4, or genetic inactivation of E2F1, results in decreased expression of Kir6.2, impaired insulin secretion and glucose intolerance in mice. Furthermore we show that rescue of Kir6.2 expression restores insulin secretion in E2f1(-/-) beta cells. Finally, we demonstrate that CDK4 is activated by glucose through the insulin pathway, ultimately resulting in E2F1 activation and, consequently, increased expression of Kir6.2. In summary we provide evidence that the CDK4-pRB-E2F1 regulatory pathway is involved in glucose homeostasis, defining a new link between cell proliferation and metabolism.

CXC ligand 5 is an adipose-tissue derived factor that links obesity to insulin resistance.

We show here high levels of expression and secretion of the chemokine CXC ligand 5 (CXCL5) in the macrophage fraction of white adipose tissue (WAT). Moreover, we find that CXCL5 is dramatically increased in serum of human obese compared to lean subjects. Conversely, CXCL5 concentration is decreased in obese subjects after a weight reduction program, or in obese non-insulin-resistant, compared to insulin-resistant, subjects. Most importantly we demonstrate that treatment with recombinant CXCL5 blocks insulin-stimulated glucose uptake in muscle in mice. CXCL5 blocks insulin signaling by activating the Jak2/STAT5/SOCS2 pathway. Finally, by treating obese, insulin-resistant mice with either anti-CXCL5 neutralizing antibodies or antagonists of CXCR2, which is the CXCL5 receptor, we demonstrate that CXCL5 mediates insulin resistance. Furthermore CXCR2-/- mice are protected against obesity-induced insulin resistance. Taken together, these results show that secretion of CXCL5 by WAT resident macrophages represents a link between obesity, inflammation, and insulin resistance.

The nuclear receptor liver receptor homolog-1 is an estrogen receptor target gene.

Liver receptor homolog-1 (LRH-1) is a nuclear receptor previously known to have distinct functions during mouse development and essential roles in cholesterol homeostasis. Recently, a new role for LRH-1 has been discovered in tumor progression, giving LRH-1 potential transforming functions. In order to identify critical factors stimulating LRH-1 expression leading to deregulated cellular proliferation, we studied its expression and its regulation in several breast cancer cell lines. We observed that LRH-1 expression was increased in estrogen receptor (ER) alpha expressing cell lines, whereas weak-to-no expression was found in nonexpressing ERalpha cell lines. In MCF7, LRH-1 expression was highly induced after treatment with 17beta-estradiol (E2). This transcriptional regulation was the result of a direct binding of the ER to the LRH-1 promoter, as demonstrated by gelshift and chromatin immunoprecipitation assays. Interestingly, siRNA-mediated inactivation of LRH-1 decreased the E2-dependent proliferation of MCF7 cells. Finally, LRH-1 protein expression was detected by immunohistochemistry in tumor cells of human mammary ductal carcinomas. Altogether, these data demonstrate that LRH-1 is transcriptionally regulated by the ER alpha and reinforce the hypothesis that LRH-1 could exert potential oncogenic effects during breast cancer formation.

The intramacrophagic environment of Brucella suis and bacterial response.

Phagocytes have developed various antimicrobial defense mechanisms to eliminate pathogens. They comprise the oxidative burst, acidification of phagosomes, or fusion of phagosomes with lysosomes. Facultative intracellular bacteria, in return, have developed strategies counteracting the host cell defense, resulting in intramacrophagic survival. Until lately, only very little was known about the phagosomal compartment containing Brucella spp., the environmental conditions the bacteria encounter, and the pathogen's stress response. Recently, we have determined that the phagosomes acidify rapidly to a pH of 4.0-4.5 following infection, but this early acidification is crucial for intracellular replication as neutralization results in bacterial elimination. A vacuolar proton-ATPase is responsible for this phenomenon that is not linked to phagosome-lysosome fusion. On the contrary, in vitro reconstitution assays revealed association only between phagosomes containing killed B. suis and lysosomes, describing the absence of phagolysosome fusion due to specific recognition inhibition for live bacteria. Further evidence for the necessity of an intact, acidic phagosome as a predominant niche of brucellae in macrophages was obtained with a strain of B. suis secreting listeriolysin. It partially disrupts the phagosomal membranes and fails to multiply intracellularly. How does B. suis adapt to this environment? We have identified and studied a series of genes that are involved in this process of adaptation. The bacterial heat shock protein and chaperone DnaK is induced in phagocytes and it is essential for intracellular multiplication. A low-level, constitutive expression of dnaK following promoter exchange does not restore intramacrophagic survival. Another chaperone and heat shock protein, ClpB, belonging to the family of ClpATPases, is important for the resistance of B. suis to several in vitro stresses, but does not contribute to intramacrophagic survival of the pathogen. Additional bacterial genes specifically induced within the phagocyte were identified by an intramacrophagic screen of random promoter fusions to the reporter gene gfp. A large majority of these genes are encoding proteins involved in transport of nutrients (sugars, amino acids), or cofactors, such as nickel. Analysis of the intracellular gene activation reveals that low oxygen tension is encountered by B. suis. Altogether, these results suggest three major stress conditions encountered by brucellae in the phagosome: acid stress, starvation and low oxygen tension.

The analysis of the intramacrophagic virulome of Brucella suis deciphers the environment encountered by the pathogen inside the macrophage host cell.

The pathogen Brucella suis resides and multiplies within a phagocytic vacuole of its host cell, the macrophage. The resulting complex relationship has been investigated by the analysis of the set of genes required for virulence, which we call intramacrophagic virulome. Ten thousand two hundred and seventy-two miniTn5 mutants of B. suis constitutively expressing gfp were screened by fluorescence microscopy for lack of intracellular multiplication in human macrophages. One hundred thirty-one such mutants affected in 59 different genes could be isolated, and a function was ascribed to 53 of them. We identified genes involved in (i) global adaptation to the intracellular environment, (ii) amino acid, and (iii) nucleotide synthesis, (iv) sugar metabolism, (v) oxidoreduction, (vi) nitrogen metabolism, (vii) regulation, (viii) disulphide bond formation, and (ix) lipopolysaccharide biosynthesis. Results led to the conclusion that the replicative compartment of B. suis is poor in nutrients and characterized by low oxygen tension, and that nitrate may be used for anaerobic respiration. Intramacrophagic virulome analysis hence allowed the description of the nature of the replicative vacuole of the pathogen in the macrophage and extended our understanding of the niche in which B. suis resides. We propose calling this specific compartment "brucellosome."

Induction of dnaK through its native heat shock promoter is necessary for intramacrophagic replication of Brucella suis.

The heat shock protein DnaK is essential for intramacrophagic replication of Brucella suis. The replacement of the stress-inducible, native dnaK promoter of B. suis by the promoter of the constitutively expressed bla gene resulted in temperature-independent synthesis of DnaK. In contrast to a dnaK null mutant, this strain grew at 37 degrees C, with a thermal cutoff at 39 degrees C. However, the constitutive dnaK mutant, which showed high sensitivity to H(2)O(2)-mediated stress, failed to multiply in murine macrophage-like cells and was rapidly eliminated in a mouse model of infection, adding strong arguments to our hypothesis that stress-mediated and heat shock promoter-dependent induction of dnaK is a crucial event in the intracellular replication of B. suis.

Functional analysis of the ClpATPase ClpA of Brucella suis, and persistence of a knockout mutant in BALB/c mice.

The protein ClpA belongs to a diverse group of polypeptides named ClpATPases, which are highly conserved, and which include several molecular chaperones. In this study the gene encoding the 91 kDa protein b-ClpA of the facultative intracellular pathogen Brucella suis, which showed 70% identity to ClpA of Rhodobacter blasticus, was identified and sequenced. Following heterologous expression in Escherichia coli strains SG1126 (DeltaclpA) and SG1127 (Deltalon DeltaclpA), b-ClpA replaced the function of E. coli ClpA, participating in the degradation of abnormal proteins. A b-clpA null mutant of B. suis was constructed, and growth experiments at 37 and 42 degrees C showed reduced growth rates for the null mutant, especially at the elevated temperature. The mutant complemented by b-clpA and overexpressing the gene was even more impaired at 37 and 42 degrees C. In intracellular infection of human THP-1 or murine J774 macrophage-like cells, the clpA null mutant and, to a lesser extent, the strain of B. suis overexpressing b-clpA behaved similarly to the wild-type strain. In a murine model of infection, however, the absence of ClpA significantly increased persistence of B. suis. These results showed that in B. suis the highly conserved protein ClpA by itself was dispensable for intramacrophagic growth, but was involved in temperature-dependent growth regulation, and in bacterial clearance from infected BALB/c mice.