Cdk4 is indispensable for postnatal proliferation of the anterior pituitary

For proper development and tissue homeostasis, cell cycle progression is controlled by multilayered mechanisms. Recent studies using knock-out mice have shown that animals can develop relatively normally with deficiency for each of the G1/S-regulatory proteins, D-type and E-type cyclins, cyclin-dependent kinase 4 (Cdk4), and Cdk2. Although Cdk4-null mice show no embryonic lethality, they exhibit specific endocrine phenotypes, i.e. dwarfism, infertility, and diabetes. Here we have demonstrated that Cdk4 plays an essential non-redundant role in postnatal proliferation of the anterior pituitary. Pituitaries from wild-type and Cdk4-null embryos at embryonic day 17.5 are morphologically indistinguishable with similar numbers of cells expressing a proliferating marker, Ki67, and cells expressing a differentiation marker, growth hormone. In contrast, anterior pituitaries of Cdk4-null mice at postnatal 8 weeks are extremely hypoplastic with markedly decreased numbers of Ki67+ cells, suggesting impaired cell proliferation. Pituitary hyperplasia induced by transgenic expression of human growth hormone-releasing hormone (GHRH) is significantly diminished in the Cdk4+/- genetic background and completely abrogated in the Cdk4-/- background. Small interfering RNA (siRNA)-mediated knockdown of Cdk4 inhibits GHRH-induced proliferation of GH3 somato/lactotroph cells with restored expression of GHRH receptors. Cdk4 siRNA also inhibits estrogen-dependent cell proliferation in GH3 cells and closely related GH4 cells. In contrast, Cdk6 siRNA does not diminish proliferation of these cells. Furthermore, Cdk4 siRNA does not affect GHRH-induced proliferation of mouse embryonic fibroblasts or estrogen-dependent proliferation of mammary carcinoma MCF-7 cells. Taken together, Cdk4 is dispensable for prenatal development of the pituitary or proliferation of other non-endocrine tissues but indispensable specifically for postnatal proliferation of somato/lactotrophs.     

Identification and characterization of a novel component of the cornified envelope, cornifelin

Psoriasis is recognized as a chronic inflammatory disease characterized by epidermal hyperproliferation. To identify psoriasis-related genes, we compared the mRNA populations of normal and psoriatic skin. We identified one gene, designated as cornifelin, which showed increased expression in psoriatic skin. Human cornifelin contains 112 amino acids and is expressed in the uterus, cervix, and skin. In situ hybridization analysis demonstrated the presence of human cornifelin in the granular cell layer of the epidermis. To investigate the function of cornifelin, we established a transgenic mouse line overexpressing human cornifelin. Using these mice, we have shown that cornifelin is directly or indirectly cross-linked to at least two other cornified envelope proteins, loricrin and involucrin, in vivo. Overexpression of human cornifelin correlated with decreased loricrin expression and increased involucrin expression in the transgenic mouse. However, abnormality of epidermal differentiation was not observed in the transgenic mouse.     

Flavonoid-induced reduction of ENaC expression in the kidney of Dahl salt-sensitive hypertensive rat

Flavonoid, a plant extract, exhibits various biological actions. Dietary flavonoid intake is reported to reduce an elevated blood pressure, however the mechanism is unknown. The epithelial Na+ channel (ENaC) in the kidney plays a key role in the regulation of blood pressure by contributing to the Na+ reabsorption in renal tubules. Thus, we investigated the effect of quercetin, a flavonoid, on ENaC mRNA expression in the kidney of hypertensive Dahl salt-sensitive rats. Dahl salt-sensitive rats of 8 weeks were acclimated for 1 week in a metabolic cage and were subsequently kept for 4 weeks under four different conditions: (1) normal salt diet (0.3% NaCl), (2) normal salt diet with quercetin (10 mg/kg/day), (3) high-salt diet (8% NaCl), and (4) high-salt diet with quercetin. Quercetin diminished the alphaENaC mRNA expression in the kidney associated with reduction of the systolic blood pressure elevated by high-salt diet, suggesting that one of the mechanisms of the flavonoid's antihypertensive effect on salt-sensitive hypertension would be mediated through downregulation of ENaC expression in the kidney.     

Prostanoid EP4 receptor is involved in suppression of 3T3-L1 adipocyte differentiation

Prostaglandins (PGs) have been shown to play various roles in adipogenesis. In this study, we investigated on which PGE receptor subtypes are involved in the inhibition of 3T3-L1 preadipocyte differentiation. The triglyceride content of cells, used as an index of differentiation, was decreased when PGE(2), the FP-agonist fluprostenol or dibutyryl cAMP, was exogenously added to differentiation cocktails. 3T3-L1 preadipocyte cells express mRNAs for the prostanoid EP4, FP, and IP receptors. PGE(2) and the EP4 agonist AE1-329 increased cAMP levels in preadipocytes in a dose-dependent manner. AE1-329 suppressed the expression induction of differentiation marker genes such as resistin and peroxisome proliferator-activated receptor-gamma. The inhibitory effect of PGE(2) but not that of fluprostenol was reversed by the addition of the EP4 antagonist AE3-208. AE3-208 mimicked the differentiation-promoting effects of indomethacin. These results suggest that the EP4 receptor mediates the suppressive action of PGE(2) in 3T3-L1 adipocyte differentiation.     

Leptin and insulin down-regulate angiopoietin-like protein 3, a plasma triglyceride-increasing factor

We reported previously that angiopoietin-like protein3 (ANGPTL3), a liver-specific secretory factor, increased plasma triglyceride (TG) via inhibition of lipoprotein lipase and free fatty acid (FFA) by activating adipose-lipolysis. The current study examined the regulation of Angptl3 by leptin and insulin, both of which are key players in the metabolic syndrome. Angptl3 expression and plasma ANGPTL3 levels were increased in leptin-resistant C57BL/6J(db/db) and -deficient C57BL/6J(ob/ob) mice, relative to the control. Leptin supplements decreased Angptl3 gene expression and plasma ANGPTL3 in C57BL/6J(ob/ob) mice. The changes of Angptl3 were associated with alterations of plasma TG and FFA levels. Leptin treatment directly suppressed Angptl3 gene expression in hepatocytes. Angptl3 gene expression and plasma protein levels were also increased in insulin-deficient streptozotocin-treated mice. Insulin treatment of hepatocytes decreased Angptl3 gene expression and protein secretion. Our results suggest that elevated ANGPTL3 by leptin- or insulin-resistance is attributed to increased plasma TG and FFA concentrations in obesity.     

WGEF is a novel RhoGEF expressed in intestine, liver, heart, and kidney

Rho family GTPases regulate multiple cellular processes through their downstream effectors, where their activities are stimulated by the guanine nucleotide exchange factors. Here, we report a new member of RhoGEF, WGEF, which has the classical structure of DH-PH domain and a C-terminal SH3 domain. WGEF was shown to activate RhoA, Cdc42, and Rac1 by pulldown assay, and forced expression of WGEF resulted in marked rearrangement of the actin cytoskeleton, which is typically seen by the activation of RhoA, Cdc42, and Rac1. WGEF was highly expressed in intestine and also in liver, heart and kidney, which may suggest the involvement of WGEF in the development and functions of these organs. The expression pattern may also suggest the possible importance of WGEF in the understanding of diseases based on metabolic disorder.     

Domain architecture and activity of human Pex19p, a chaperone-like protein for intracellular trafficking of peroxisomal membrane proteins

Pex19p is a peroxin involved in peroxisomal membrane biogenesis and probably functions as a chaperone and/or soluble receptor specific for cargo peroxisomal membrane proteins (PMPs). To elucidate the functional constituents of Pex19p in terms of the protein structure, we investigated its domain architecture and binding affinity toward various PMPs and peroxins. The human Pex19p cDNA was overexpressed in Escherichia coli, and a highly purified sample of the Pex19p protein was prepared. When PMP22 was synthesized by cell-free translation in the presence of Pex19p, the PMP22 bound to Pex19p was soluble, whereas PMP22 alone was insoluble. This observation shows that Pex19p plays a role in capturing PMP and maintaining its solubility. In a similar manner, Pex19p was bound to PMP70 and Pex16p as well as the Pex3p soluble fragment. Limited proteolysis analyses revealed that Pex19p consists of the C-terminal core domain flanking the flexible N-terminal region. Separation of Pex19p into its N- and C-terminal halves abolished interactions with PMP22, PMP70, and Pex16p. In contrast, the flexible N-terminal half of Pex19p was bound to the Pex3p soluble fragment, suggesting that the binding mode of Pex3p toward Pex19p differs from that of other PMPs. This idea is supported by our detection of the Pex19p-Pex3p-PMP22 ternary complex.