Sumiya Eto

Androgen receptors, 5 alpha-reductase activity and androgen-dependent proliferation of vascular smooth muscle cells

To assess the direct effect of androgen on the development of atherosclerosis, we investigated the effect of androgen and its receptor expression in rat vascular smooth muscle cells (VSMC) isolated from rat aorta. We detected the androgen receptor mRNA in VSMC by reverse transcription of the total RNA coupled with amplification of the resulting cDNA by polymerase chain reaction. Binding studies revealed the presence of a single class of binding sites for testosterone (Kd 7.37 nM, Bmax 10.59 fmol/mg protein) and dihydrotestosterone (DHT; Kd 4.89 nM, Bmax 11.37 fmol/mg protein) in VSMC. Measurement of 5 alpha-reductase activity suggested that testosterone is converted to DHT in VSMC (Km 0.36 microM, Vmax 623 fmol/mg protein/h). Moreover, in the present study, DHT significantly stimulated DNA synthesis of VSMC (120-160% of control). The mitogenic activity of testosterone is much less potent than that of DHT. Considering these results, we concluded that androgen may directly accelerate atherosclerosis by stimulating the proliferation of VSMC.

Interleukin-4 as a potent inhibitor of bone resorption

A possible role of interleukin-4 (IL-4) in the regulation of bone turnover was assessed by employing a 45Ca prelabeled-fetal mouse long bone culture system. IL-4 inhibited the bone resorption stimulated by parathyroid hormone (PTH), PTH related protein (PTHrP), 1 alpha, 25, dihydroxy-vitamin D3 [1 alpha, 25 (OH)2 D3], interleukin-1 alpha and - 1 beta (IL-1 alpha, IL-1 beta) and prostaglandin E2 (PGE2). Anti-IL-4 on monoclonal antibody abolished the inhibitory effect of IL-4 on the bone resorption. These results suggest that IL-4 may play an important role on the inhibitory regulation of bone resorption.

Chondrocytes Are Regulated by Cellular Adhesion Through CD44 and Hyaluronic Acid Pathway

The articular cartilage consists of resident chondrocytes embedded within the extracellular matrix which contains several components such as collagen and hyaluronic acids (HA). CD44 is a major cell surface receptor for HA and is homologous to cartilage‐link proteins. Although CD44 is present in cartilage, it is not clear if chondrocytes adhere to HA through CD44 or whether such adhesion changes the function of chondrocytes. We studied the molecular mechanisms of CD44‐related chondrocyte adhesion to HA and the effects of such adhesion on chondrocyte function. Experiments were performed using the human chondrosarcoma‐derived chondrocyte‐like cell line HCS‐2/8. Our results showed that (a) HCS‐2/8 cells highly expressed CD44; (b) HCS‐2/8 cells efficiently adhered to HA without any stimuli; (c) monoclonal antibody (mAb)‐blocking studies indicated that adhesion of HCS‐2/8 cells to HA was mainly mediated by the CD44/HA pathway; (d) cellular adhesion to HA increased the proliferation of HCS‐2/8 cells, independent of transforming growth factor‐β (TGF‐β), but this was inhibited by CD44 mAb; (e) the adhesion of chondrocytes to HA also induced c‐myc mRNA expression and this was also inhibited by CD44 mAb; and (f) the adhesion of cells to HA augmented TGF‐β mRNA expression, a process also reduced by CD44 mAb. Thus, HCS‐2/8 cells effectively adhered to HA through cell surface CD44. The adhesion was also involved in cellular signaling which induced cellular proliferation and expression of c‐myc mRNA as well as TGF‐β mRNA expression within the cells. Our results indicate that CD44 on chondrocytes plays an important role in normal and abnormal functions of cartilage through its adhesion to HA, which induces a variety of stimulatory signals to regulate chondrocyte proliferation as well as matrix synthesis in cartilage microenvironment.

Intercellular Adhesion Molecule 1 Discriminates Functionally Different Populations of Human Osteoblasts: Characteristic Involvement of Cell Cycle Regulators

The concept of differential regulation of certain adhesion molecules on different cell subsets and their relevance to cell functions has emerged in recent years. The initial event in bone remodeling is an increase in osteoclastic bone resorption and cell adhesion between osteoclastic precursors and bone marrow stromal cells or osteoblasts is known to commit the osteoclast development. Here, we show that human osteoblasts can be divided into two subsets based on the expression of the intercellular adhesion molecule (ICAM)‐1; ICAM‐1+ osteoblasts highly adhered to monocytes, including osteoclast precursors, produced osteoclast differentiation factor (ODF), and induced multinuclear osteoclast‐like cell formation. Anti‐ODF monoclonal antibody (mAb) did not inhibit the adhesion of monocytes to osteoblastic cells, whereas anti‐leukocyte function‐associated antigen (LFA)‐1, a receptor for ICAM‐1, mAb blocked the adhesion. We thereby propose that the higher affinity adhesion via LFA‐1/ICAM‐1 is prerequisite for efficient function of membrane‐bound ODF during osteoclast maturation. The functional characteristics of ICAM‐1+ osteoblasts were emphasized further by cell cycle regulation, as manifested by (i) up‐regulation of p53 and p21, (ii) reduction of activity of cyclin‐dependent kinase (cdk) 6, (iii) underphosphorylation of retinoblastoma protein, (iv) increased Fas but reduced bcl‐2 expression, and (v) majority of cells remained at G0/G1 phase. Furthermore, ICAM‐1+ osteoblasts were induced by interleukin‐1β (IL‐1β). Taken together, we propose that the differentiation of osteoblasts to ICAM‐1+ subpopulation by inflammatory cytokines plays an important role in osteoporosis, which is observed in patients with chronic inflammation, because ICAM‐1+ osteoblasts can bias bone turnover to bone resorption, committing osteoclast maturation through cell adhesion with its precursor, and the majority of ICAM‐1+ osteoblasts arrested at G0/G1 phase. Such regulation of cell cycle arrest also is an important determinant of the life span of cells in bone in which continuous bone remodeling maintains its homeostasis.

Centrally Administered Murine-Leptin Stimulates the Hypothalamus-Pituitary- Adrenal Axis through Arginine-Vasopressin

Starvation induces a decrease in circulating leptin levels and activation of the hypothalamus-pituitary-adrenal (HPA) axis. Leptin inhibits the HPA axis in unfed rodents or genetically leptin-deficient ob/ob mice, whereas it stimulates corticotropin-releasing hormone (CRH) gene expression in the paraventricular nucleus (PVN). However, the interactions between leptin, CRH and the HPA axis are poorly understood and are likely to be complex. We recently demonstrated that central leptin administration caused increases in plasma arginine-vasopressin (AVP) and AVP gene expression of the PVN in nonstressful rats. AVP stimulates the release of adrenocorticotropic hormone (ACTH), but it also potentiates the action of CRH on ACTH release. In this study, we investigated the effects of leptin on plasma ACTH and corticosterone levels, CRH mRNA of the PVN and proopiomelanocortin (POMC) mRNA of the pituitary in nonstrained rats. Intracerebroventricularly administered leptin caused increases in plasma ACTH and corticosterone levels in dose-dependent manners. In Northern blot analyses, the leptin injection induced significant increases in the expression of CRH mRNA in the PVN and POMC mRNA in the pituitary. The increased plasma ACTH and corticosterone levels by leptin were attenuated with intracerebroventricular pretreatment of a V1a receptor antagonist (OPC-21268) or a V1a/V1b receptor antagonist (dP[Tyr(Me)2]AVP), but not with that of a V2 receptor antagonist (OPC-31260). The leptin-induced CRH mRNA expression in the PVN and POMC mRNA expression in the pituitary were also reduced by the pretreatment with OPC-21268 and dP[Tyr(Me)2]AVP. These results suggest that intracerebroventricular leptin administration activates the HPA axis by AVP receptor activation through V1a receptors in the PVN which in turn activates CRH neurons to drive ACTH and corticosterone secretion in concert with AVP in nonstrained rats.

The receptor, metabolism and effects of androgen in osteoblastic MC3T3-E1 cells

We investigated the androgen receptor (AR), metabolism and effects of androgens in osteoblastic MC3T3-E1 cells. AR was proved as a transcript of a 10-kb mRNA and as a 110-kDa protein. An immunocytochemical study showed that AR was located mainly in the nuclei. Specific binding of [3H]DHT was observed in both the nuclear and cytosol fractions. MC3T3-E1 cells possessed approximately 1190 binding sites per cell and most of the sites (1150 sites) situated in the nucleus. The apparent Kd value in the nuclear fraction was 1.35 nM for [3H]DHT binding, and it was similar to that for [3H]testosterone. In the competition analysis, there was not much difference in the displacement of the [3H]DHT binding from AR between the addition of radioinert DHT and testosterone. In studies of 5 alpha-reductase activity and aromatase activity of the cells, both activities were lower than the respective values in classical androgen target tissues. Androgens stimulated the incorporation of [3H]thymidine into the cell, and DHT and testosterone had a similar potency on the cell proliferation. Thus, these results suggest testosterone itself acts mainly on the osteoblasts without conversion to DHT.

Parathyroid Hormone-Related Peptide-(1–34) [PTHrP- (1–34)] Induces Vasopressin Release from the Rat Supraoptic Nucleus in Vitro through a Novel Receptor Distinct from a Type I or Type II PTH/PTHrP Receptor

PTH and PTH-related peptide (PTHrP) bind to a type I PTH/PTHrP receptor expressed in bone and kidney or a type II receptor in nonclassical target tissue with equal affinity and similar bioactivities. PTHrP is abundant in the central nervous system, but its physiological role remains unknown. Herein, we examined the role of PTHrP-(1–34) on arginine vasopressin (AVP) release from the rat supraoptic nucleus (SON). Application of PTHrP-(1–34) to SON slices caused an increase in AVP release in a concentration-dependent manner. Neither PTHrP-(7–34) nor PTH-(1–34) had any effect on AVP release from the SON. PTHrP-(1–34)-induced AVP release was antagonized by a large excess of PTHrP-(7–34) and by H89, an inhibitor of cAMP-dependent protein kinase (A kinase), but not by PTH-(1–34) or PTH-(13–34). PTHrP-(1–34), but not PTH-(1–34), also dose-dependently increased the levels of cAMP in the SON. 125I-Labeled PTHrP-(1–34) bound specifically to crude membranes isolated from the SON. Scatchard analysis showed a single cl...

Effect of hyperbaric oxygenation on macrophage function in mice

Hyperbaric oxygenation (HBO) has an immunosuppressive effect. The possible mechanisms of this immunosuppressive effect were assessed by determining the production of interleukin-1 (IL-1), interleukin-6 (IL-6), and prostaglandin E2 (PGE2), as well as phagocytosis by splenic macrophages from HBO-treated mice. Although HBO treatment did not have any significant effect on IL-6 production and phagocytotic activity, a marked decrease in IL-1 production and a significant decrease in PGE2 production were observed. These results suggest that the reduction of IL-1 production may play an important role in the immunosuppressive effect of HBO.

Effect of interleukin 1 (IL-1) on the levels of cytochrome P-450 involving IL-1 receptor on the isolated hepatocytes of rat

In the present study, the expression of IL-1 receptor (IL-1 R) on the primary cultured hepatocytes was determined and the effect of IL-1 on the intracellular amount of cytochrome P-450 was investigated. The kinetics of IL-1R on the hepatocytes was studied by [125I]-IL-1 alpha and revealed that 5,000 molecules of IL-1 bound to a hepatocyte with a Kd of 4.0 x 10(-9) M. Incubation of the hepatocytes with IL-1 for 18 hrs decreased the contents of cytochrome P-450 in a dose-dependent manner. These findings suggest that IL-1 potentially depresses the drug metabolism involving IL-1R which is expressed on hepatocytes.

Short-Term Treatment of Recombinant Murine Interleukin-4 Rapidly Inhibits Bone Formation in Normal and Ovariectomized Mice

Estrogen deficiency contributes to an increase in bone resorption and bone formation characterized by a high rate of bone turnover. Interleukin-4 (IL-4) is a rapid and potent inhibitor of bone resorption. We examined the short term in vivo effects of recombinant murine IL-4 (rmIL-4) on bone remodeling in normal and ovariectomized mice. Eight-week-old mice were randomized into the following five groups: (1) sham-operated mice (sham); (2) sham-operated mice infused with rmIL-4; (3) ovariectomized mice (ovx); (4) ovx infused with rmIL-4; and (5) ovx replaced by 10 or 20 microg of 17beta-estradiol (E2) for 14 or 28 days after ovariectomy, respectively. rmIL-4 at a dose of 5 microg/day was infused into ovx and sham for 3 days prior to sacrifice. Analyses were performed 14 and 28 days after operation. An increase in serum alkaline phosphatase and urinary deoxypyridinoline levels induced by ovariectomy was inhibited by the 3-day infusion of rmIL-4. In ovx, serum and urinary IL-6 levels were also increased significantly 14 days after ovariectomy, which were restored by E2 but not by rmIL-4. Histomorphometrical analysis of trabecular bone revealed that the 3-day infusion of rmIL-4 inhibited the high rate of bone turnover induced by ovariectomy, such as an increase in the osteoclastic surface (Oc.S/BS), number of osteoclasts per mm bone surface (N.Oc/BS), mineralized surface per mm bone surface (MS/BS), and bone mineral apposition rate (MAR). A significant decrease in the bone volume (BV/TV) observed in ovx was not modulated by a 3-day infusion of rmIL-4 prior to sacrifice. In sham, rmIL-4 also caused a significant decrease in the Oc.S/BS, N.Oc/BS, MS/BS, and MAR, but the BV/TV was not modulated by rmIL-4. We conclude that short term infusion of rmIL-4 in vivo rapidly inhibits not only bone resorption but also its formation in both sham-operated and ovariectomized growing mice, resulting in a low rate of bone turnover without modulating bone volume.