Constitutively active parathyroid hormone receptor signaling in cells in osteoblastic lineage suppresses mechanical unloading-induced bone resorption

Multiple signaling pathways participate in the regulation of bone remodeling, and pathological negative balance in the regulation results in osteoporosis. However, interactions of signaling pathways that act comprehensively in concert to maintain bone mass are not fully understood. We investigated roles of parathyroid hormone receptor (PTH/PTHrP receptor) signaling in osteoblasts in unloading-induced bone loss using transgenic mice. Hind limb unloading by tail suspension reduced bone mass in wild-type mice. In contrast, signaling by constitutively active PTH/PTHrP receptor (caPPR), whose expression was regulated by the osteoblast-specific Col1a1 promoter (Col1a1-caPPR), suppressed unloading-induced reduction in bone mass in these transgenic mice. In Col1a1-caPPR transgenic (Tg) mice, hind limb unloading suppressed bone formation parameters in vivo and mineralized nodule formation in vitro similarly to those observed in wild-type mice. In addition, serum osteocalcin levels and mRNA expression levels of type I collagen, Runx2 and Osterix in bone were suppressed by unloading in both wild-type mice and Tg mice. However, in contrast to unloading-induced enhancement of bone resorption parameters in wild-type mice, Col1a1-caPPR signaling suppressed, rather than enhanced, osteoclast number and osteoclast surface as well as urinary deoxypyridinoline excretion upon unloading. Col1a1-caPPR signaling also suppressed mRNA expression levels of RANK and c-fms in bone upon unloading. Although the M-CSF and monocyte chemoattractant protein 1 (MCP-1) mRNA levels were enhanced in control Tg mice, these levels were suppressed in unloaded Tg mice. These results indicated that constitutive activation of PTH/PTHrP receptor signaling in osteoblastic cells suppresses unloading-induced bone loss specifically through the regulation of osteoclastic activity.     

Mercuric chloride mediates a protein sulfhydryl modification-based pathway of signal transduction for activating Src kinase which is independent of the phosphorylation / dephosphorylation of a carboxyl terminal tyrosine

Little is known about the regulatory mechanism of c-Src kinase in cells except the suggested regulation through phosphorylation and dephosphorylation of its carboxyl terminal tyrosine residue (Y527). We here demonstrated that exposure of NIH3T3 cells to mercuric chloride (HgCl₂) induces both aggregation and activation of Src kinase protein through a redox-linked mechanism. The aggregation of Src proteins was suggested to be induced by the sulfhydryl groups-to-Hg²⁺ reaction-mediated polymerization of cell membrane proteins to which the Src proteins associate noncovalently. The possibility was ruled out that the aggregation occurred secondarily to the promotion of protein tyrosine phosphorylation. Further study revealed that the Src kinase was activated by HgCl₂ at least in part independent of the known Csk kinase-linked or Y527-phosphorylation/dephosphorylation-mediated control. Correspondingly, CNBr cleavage mapping of phosphopeptides for autophosphorylated c-Src protein demonstrated selective promotion of phosphorylation at Y416 in HgCl₂-treated cells without obvious change in the phosphorylation level at Y527. These results suggest a unique protein sulfhydryl modification-based pathway of signal transduction for activating Src kinase in NIH3T3 cells. © 1996 Wiley-Liss, Inc.     

Role of PKC and TGF-beta receptor in glucose-induced proliferation of smooth muscle cells

The role of protein kinase C (PKC) and transforming growth factor (TGF)-beta in the proliferation of vascular smooth muscle cells (SMCs) under a high glucose condition was investigated. [3H]-thymidine incorporation under 20 mM glucose was significantly accelerated compared with that under 5.5 mM glucose, and this increase was inhibited by an anti-TGF-beta antibody or a PKC-beta specific inhibitor, LY333531. The amount of active and total TGF-beta1 in the conditioned media did not differ between 5.5 and 20 mM glucose. However, the expression of TGF-beta receptor type II under 20 mM glucose was significantly increased, but that of the TGF-beta receptor type I was not. This increased expression of the TGF-beta receptor type II was prevented by LY333531. These observations suggest that the increased expression of the TGF-beta receptor type II via PKC-beta plays an important role in the accelerated proliferation of SMCs under a high glucose condition, leading to the development of diabetic macroangiopathy.     

Inhibitory effects of fluvastain and its metabolites on the formation of several reactive oxygen species

We investigated the inhibitory effects of fluvastain (FV) and its metabolites (M-2, M-3, M-4, M-5, and M-7) on the formation of several reactive oxygen species (ROS), such as singlet oxygen (1O2), superoxide anion (O2-), hydroxy radical (*OH), hypochlorite ion (OCL-), and linoleic acid peroxide (LOO*). Inhibitory effects of pravastatin (PV), simvastatin (SV), probucol (PR) and alpha-tocopherol (TOC) were also tested. The inhibitory effects of 5-hydroxy FV (M-2) and 6-hydroxy FV (M-3) on the formation of 1O2, O2-, *OH, and OCL- were strongest. Scavenging of 1O2 by M-4, M-5, (+)-FV, and (-)-FV was also noted. The inhibitory effects of (+)-FV on the formation of 1O2 were comparable to those of (-)-FV, PV, SV, PR and M-7 had little or no inhibitory effect on the formation of several ROS. In conclusion, FV and its metabolites, particulary M-2 and M-3, have the potential to protect against oxidative stress mediated by several ROS.     

Structural requirements of a human deoxyribonuclease II for the development of the active enzyme form, revealed by site-directed mutagenesis

Using site-directed mutagenesis, we eliminated three potential N-glycosylation sites (N86, N212, and N266) of human deoxyribonuclease II (DNase II), conserved in mammalian enzymes, and a proteolytic processing site (Q46-R47), forming a propeptide subunit of the enzyme. We expressed a series of these mutant DNase II constructs in COS-7 and Hep G2 cells. Liberation of each glycosylation site at N86 and N266 and the cleavage site interfered dramatically with expression of the intracellular and secreted DNase II activities, irrespective of cell line transfected. A chimeric mutant in which the signal peptide of the DNase II was replaced with that of human DNase I had no intracellular or secreted enzyme activity. Therefore, a simultaneous attachment of a carbohydrate moiety to N86 and N266, cleavage of the propeptide from the single DNase II precursor, and the inherent signal peptide might be required for subcellular sorting and proteolytic maturation of the enzyme.     

Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer

Fetal liver, the major site of hematopoiesis during embryonic development, acquires additional various metabolic functions near birth. Although liver development has been characterized biologically as consisting of several distinct steps, the molecular events accompanying this process are just beginning to be characterized. In this study, we have established a novel culture system of fetal murine hepatocytes and investigated factors required for development of hepatocytes. We found that oncostatin M (OSM), an interleukin-6 family cytokine, in combination with glucocorticoid, induced maturation of hepatocytes as evidenced by morphological changes that closely resemble more differentiated hepatocytes, expression of hepatic differentiation markers and intracellular glycogen accumulation. Consistent with these in vitro observations, livers from mice deficient for gp130, an OSM receptor subunit, display defects in maturation of hepatocytes. Interestingly, OSM is expressed in CD45(+) hematopoietic cells in the developing liver, whereas the OSM receptor is expressed predominantly in hepatocytes. These results suggest a paracrine mechanism of hepatogenesis; blood cells, transiently expanding in the fetal liver, produce OSM to promote development of hepatocytes in vivo.     

Effect of high-NaCl or high-KCl diet on hepatic Na+- and K+-receptor sensitivity and NKCC1 expression in rats

We previously reported that the bumetanide-sensitive Na(+)-K(+)-2Cl- cotransporter (NKCC1) is involved in the hepatic Na+ and K+ sensor mechanism. In the present study, we examined the effects of a high-NaCl or high-KCl diet on hepatic Na+ and K+ receptor sensitivity and NKCC1 expression in the liver of Sprague-Dawley rats. RT-PCR and Western blots were used to measure NKCC1 mRNA and protein expression, respectively. Infusion of hypertonic NaCl or isotonic KCl + NaCl solutions into the portal vein increased hepatic afferent nerve activity (HANA) in a Na+ or K+ dose-dependent manner. After 4 wk on a high-NaCl or high-KCl diet, HANA responses were attenuated compared with animals fed a normal diet, and NKCC1 expression was reduced. These results show that a high-NaCl or high-KCl diet decreases NKCC1 expression in the liver, and it might cause a reduction in hepatic Na(+)- and K(+)-receptor sensitivity.