Hiroki Kondou

PTH/cAMP/PKA Signaling Facilitates Canonical Wnt Signaling Via Inactivation of Glycogen Synthase Kinase-3β in Osteoblastic Saos-2 Cells

Although the intermittent administration of PTH is known to stimulate the bone formation, the underlying mechanisms are not fully understood. Here we investigated the crosstalk between PTH/cAMP signaling and canonical Wnt signaling using the human osteoblastic cell line Saos‐2. Treatment with PTH or forskolin, an activator of adenylate cyclase, facilitated T‐cell factor (TCF)‐dependent transactivation in a dose‐dependent manner, which was abolished by pre‐treatment with a PKA inhibitor, H89. Wnt3a and forskolin synergistically increased the TCF‐dependent transactivation. Interestingly, intermittent treatment with PTH enhanced the TCF‐dependent transactivation more profoundly than continuous treatment. In addition to the effects on TCF‐dependent reporter activity, treatment with PTH or forskolin resulted in the increased expression of endogenous targets of Wnts, Wnt‐induced secreted protein 2 (WISP2) and naked cuticle 2 (NKD2). We then investigated the convergence point of PTH/cAMP signaling and the canonical Wnt pathway. Western blotting demonstrated that GSK‐3β was rapidly phosphorylated at Ser9 on treatment with PTH or forskolin, leading to its inactivation. Moreover, overexpression of a constitutively active mutant of GSK‐3β abolished the TCF‐dependent transactivation induced by forskolin. On the other hand, overexpression of the Wnt antagonist Dickkopf‐1 (DKK1) failed to cancel the effects of forskolin on the canonical Wnt pathway. Interestingly, treatment with Wnt3a markedly reduced the forskolin‐induced expression of receptor activator of NF‐κB ligand (RANKL), a target gene of PTH/cAMP/PKA. These results suggest that cAMP/PKA signaling activates the canonical Wnt pathway through the inactivation of GSK‐3β, whereas Wnt signaling might inhibit bone resorption through a negative impact on RANKL expression in osteoblasts. J. Cell. Biochem. 104: 304–317, 2008.

A blocking peptide for transforming growth factor-β1 activation prevents hepatic fibrosis in vivo

BACKGROUND/AIMS Thrombospondin-1 is a major activator of transforming growth factor-beta1 (TGF-beta1), and a peptide derived from the latency-associated peptide, Leu-Ser-Lys-Leu (LSKL), inhibits the activation of TGF-beta1. In this study, the effects of LSKL on the hepatocyte damage and fibrogenesis in dimethylnitrosamine (DMN)-induced rat liver fibrosis were examined. METHODS Animals were given an intraperitoneal (i.p.) injection of DMN or saline three times per week for 4 weeks, and treated with LSKL, a control peptide, or saline i.p. daily. RESULTS Liver atrophy caused by DMN-injection was significantly inhibited in the DMN+LSKL group. The degrees of necrosis/degeneration and fibrosis scores were significantly lower in the DMN+LSKL group than in the control groups. The hydroxyproline content was significantly higher in the control groups than in the DMN+LSKL group. The amount of active TGF-beta1 was less in the DMN+LSKL group than in the control groups, and the active/total TGF-beta1 ratio in the DMN+LSKL group was suppressed in the control groups. Phosphorylation of Smad 2 in the liver was significantly decreased in the DMN+LSKL group. CONCLUSIONS The LSKL peptide prevented the progression of hepatic damage and fibrosis through the inhibition of TGF-beta1 activation and its signal transduction in vivo.

Both FGF23 and extracellular phosphate activate Raf/MEK/ERK pathway via FGF receptors in HEK293 cells

Fibroblast growth factor 23 (FGF23) is a phosphaturic hormone produced by bone and exerts its function in the target organs by binding the FGF receptor (FGFR) and Klotho. Since recent studies suggested that extracellular inorganic phosphate (Pi) itself triggers signal transduction and regulates gene expression in some cell types, we tested the notion that extracellular Pi induces signal transduction in the target cells of FGF23 also and influences its signaling, utilizing a human embryonic kidney cell line HEK293. HEK293 cells expressed low levels of klotho, and treatment with a recombinant FGF23[R179Q], a proteolysis‐resistant mutant of FGF23, resulted in phosphorylation of ERK1/2 and induction of early growth response‐1 (EGR1) expression. Interestingly, increased extracellular Pi resulted in activation of the Raf/MEK/ERK pathway and expression of EGR1, which involved type III sodium/phosphate (Na+/Pi) cotransporter PiT‐1. Since the effects of an inhibitor of Na+/Pi cotransporter on FGF23 signaling suggested that the signaling triggered by increased extracellular Pi shares the same downstream cascade as FGF23 signaling, we further investigated their convergence point. Increasing the extracellular Pi concentration resulted in the phosphorylation of FGF receptor substrate 2α (FRS2α), as did treatment with FGF23. Knockdown of FGFR1 expression diminished the phosphorylation of both FRS2α and ERK1/2 induced by the Pi. Moreover, overexpression of FGFR1 rescued the decrease in Pi‐induced phosphorylation of ERK1/2 in the cells where the expression of PiT‐1 was knocked down. These results suggest that increased extracellular Pi triggers signal transduction via PiT‐1 and FGFR and influences FGF23 signaling in HEK293 cells. J. Cell. Biochem. 111: 1210–1221, 2010.

Prospective Isolation and Characterization of Bipotent Progenitor Cells in Early Mouse Liver Development

Outgrowth of the foregut endoderm to form the liver bud is considered the initial event of liver development. Hepatic stem/progenitor cells (HSPCs) in the liver bud are postulated to migrate into septum transversum mesenchyme at around embryonic day (E) 9 in mice. The studies of liver development focused on the mid-fetal stage (E11.5-14.5) have identified HSPCs at this stage. However, the in vitro characteristics of HSPCs before E11.5 have not been elucidated. This is probably partly because purification and characterization of HSPCs in early fetal livers have not been fully established. To permit detailed phenotypic analyses of early fetal HSPC candidates, we developed a new coculture system, using mouse embryonic fibroblast cells. In this coculture system, CD13(+)Dlk(+) cells purified from mouse early fetal livers (E9.5 and E10.5) formed colonies composed of both albumin-positive hepatocytic cells and cytokeratin (CK) 19-positive cholangiocytic cells, indicating that early fetal CD13(+)Dlk(+) cells have properties of bipotent progenitor cells. Inhibition of signaling by Rho-associated coiled-coil containing protein kinase (Rock) or by nonmuscle myosin II (downstream from Rock) was necessary for effective expansion of early fetal CD13(+)Dlk(+) cells in vitro. In sorted CD13(+)Dlk(+) cells, expression of the hepatocyte marker genes albumin and α-fetoprotein increased with fetal liver age, whereas expression of CK19 and Sox17, endodermal progenitor cell markers, was highest at E9.5 but decreased dramatically thereafter. These first prospective studies of early fetal HSPC candidates demonstrate that bipotent stem/progenitor cells exist before E11.5 and implicate Rock-myosin II signaling in their development.

Humoral immunity is involved in the development of pericentral fibrosis after pediatric live donor liver transplantation

Yamada H, Kondou H, Kimura T, Ikeda K, Tachibana M, Hasegawa Y, Kiyohara Y, Ueno T, Miyoshi Y, Mushiake S, Ozono K. Humoral immunity is involved in the development of pericentral fibrosis after pediatric live donor liver transplantation.

Vinculin Functions as Regulator of Chondrogenesis

Background: Vinculin plays profound roles in cell adhesion and migration. Results: Trapping or knockdown of the vinculin gene in chondrocytes leads to impaired differentiation by affecting multiple signaling pathways. Conclusion: Vinculin has pleiotropic roles in chondrocytic differentiation. Significance: This study reveals a novel, tissue-specific function of vinculin in chondrocytes. To identify the genes involved in chondrocytic differentiation, we applied gene trap mutagenesis to a murine mesenchymal chondrogenic cell line ATDC5 and isolated a clone in which the gene encoding vinculin was trapped. The trapped allele was assumed to express a fusion protein containing a truncated vinculin lacking the tail domain and the geo product derived from the trap vector. The truncated vinculin was suggested to exert a dominant negative effect. Impaired functioning of vinculin caused by gene trapping in ATDC5 cells or knockdown in primary chondrocytes resulted in the reduced expression of chondrocyte-specific genes, including Col2a1, aggrecan, and Col10a1. The expression of Runx2 also was suppressed by the dysfunctional vinculin. On the other hand, the expression of Sox9, encoding a key transcription factor for chondrogenesis, was retained. Knockdown of vinculin in metatarsal organ cultures impaired the growth of the explants and reduced the expression of Col2a1 and aggrecan. Gene trapping or knockdown of vinculin decreased the phosphorylation of ERK1/2 but increased that of Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) and Akt during chondrocytic differentiation, suggesting a disturbance of signaling by insulin-like growth factor I (IGF-I). Knockdown of vinculin in the metatarsal organ culture abrogated the IGF-I-induced growth and inhibited the up-regulation of Col2a1 and aggrecan expression by IGF-I. Loss of vinculin function in differentiating chondrocytes impaired the activation of the p38 MAPK pathway also, suggesting its involvement in the regulation of chondrogenesis by vinculin. Our results indicate a tissue-specific function of vinculin in cartilage whereby it controls chondrocytic differentiation.

Histamine-induced inositol phosphate accumulation in type-2 astrocytes

Histamine elicited dose-dependent accumulation of [3H]inositol phosphates in type-2 astrocytes, but not in type-1 astrocytes. The ED50 was about 2.4 x 10(-6) M and the maximal response was obtained at 10(-4) M. This response was dose-dependently inhibited by H1-antagonists, mepyramine and D- and L-chlorpheniramine. Furthermore, D- and L-chlorpheniramine showed stereoselectivity in the inhibition. On the other hand, an H2-antagonist, famotidine, and an H3-antagonist, thioperamide, did not inhibit the response. These results indicate that histamine stimulates accumulation of inositol phosphates in type-2 astrocytes via H1-receptors.

Formation of inositol phosphates mediated by M3 muscarinic receptors in type-1 and type-2 astrocytes from neonatal rat cerebral cortex

Muscarinic receptor subtype in type-1 and type-2 astrocytes from rat neonalal cerebral cortex was examined for carbachol-elicited inositol phosphate (IP) formation. The formation of carbachol-elicited IP was inhibited by various muscarinic antagonists in the following relative order of potency: 4-DAMP > or = atropine >> pirenzepine > AF-DX 116. This pharmacological profile suggests that the activation of the M3 muscarinic receptor subtype is responsible for the stimulation of IP formation in both astrocytes.

Intraperitoneal Administration of Recombinant Receptor-Associated Protein Causes Phosphaturia via an Alteration in Subcellular Distribution of the Renal Sodium Phosphate Co-Transporter

Megalin is a multifunctional endocytic receptor that is expressed in renal proximal tubules and plays critical roles in the renal uptake of various proteins. It was hypothesized that megalin-dependent endocytosis might play a role in renal phosphate reabsorption. For addressing the short-term effects of altered megalin function, a recombinant protein for the soluble form of 39-kD receptor-associated protein (RAP) was administered intraperitoneally to 7-wk-old mice. Histidine (His)-tagged soluble RAP (amino acids 39 to 356) lacking the amino-terminal signal peptide and the carboxy-terminal endoplasmic reticulum retention signal was prepared by bacterial expression (designated His-sRAP). After the direct interaction between His-sRAP and megalin was confirmed, mice were given a single intraperitoneal administration of His-sRAP (3.5 mg/dose). Immunostaining and Western blot analyses demonstrated the uptake of His-sRAP and the accelerated internalization of megalin in proximal tubular cells 1 h after administration. In addition, internalization of the type II sodium/phosphate co-transporter (NaPi-II) was observed. The effects of three sequential administrations of His-sRAP (3.5 mg/dose, three doses at 4-h intervals) then were examined, and increased urinary excretion of low molecular weight proteins, including vitamin D-binding protein, was found, which is consistent with findings reported for megalin-deficient mice. It is interesting that urinary excretion of phosphate was also increased, and the protein level of NaPi-II in the brush border membrane was decreased. Serum concentration of 25-hydroxyvitamin D was decreased, whereas the plasma level of intact parathyroid hormone was not altered by the administration of His-sRAP. The results suggest that the His-sRAP-induced acceleration of megalin-mediated endocytosis caused phosphaturia via altered subcellular distribution of NaPi-II.

Sodium-coupled neutral amino acid transporter 4 functions as a regulator of protein synthesis during liver development.

The molecular mechanisms by which hepatocyte nuclear factor (HNF)4α regulates fetal liver development have not been fully elucidated. We screened the downstream molecules of HNF4α during liver development and identified sodium‐coupled neutral amino acid transporter (SNAT)4. The aim of this study is to investigate the regulation of SNAT4 by HNF4α and to clarify its roles in differentiating hepatocytes.