Osteoclasts, the cells that resorb bone, develop from hematopoietic precursors of the bone marrow under the control of factors produced in their microenvironment. The cytokine interleukin-6 can promote hematopoiesis and osteoclastogenesis. Interleukin-6 production by bone and marrow stromal cells is suppressed by 17 beta-estradiol in vitro. In mice, estrogen loss (ovariectomy) increased the number of colony-forming units for granulocytes and macrophages, enhanced osteoclast development in ex vivo cultures of marrow, and increased the number of osteoclasts in trabecular bone. These changes were prevented by 17 beta-estradiol or an antibody to interleukin-6. Thus, estrogen loss results in an interleukin-6-mediated stimulation of osteoclastogenesis, which suggests a mechanism for the increased bone resorption in postmenopausal osteoporosis.

Levamisole-sensitive acetylcholine receptors (L-AChRs) are ligandgated ion channels that mediate excitatory neurotransmission at the neuromuscular junctions of nematodes. They constitute a major drug target for anthelminthic treatments because they can be activated by nematode-specific cholinergic agonists such as levamisole. Genetic screens conducted in Caenorhabditis elegans for resistance to levamisole toxicity identified genes that are indispensable for the biosynthesis of L-AChRs. These include 5 genes encoding distinct AChR subunits and 3 genes coding for ancillary proteins involved in assembly and trafficking of the receptors. Despite extensive analysis of L-AChRs in vivo, pharmacological and biophysical characterization of these receptors has been greatly hampered by the absence of a heterologous expression system. Using Xenopus laevis oocytes, we were able to reconstitute functional L-AChRs by coexpressing the 5 distinct receptor subunits and the 3 ancillary proteins. Strikingly, this system recapitulates the genetic requirements for receptor expression in vivo because omission of any of these 8 genes dramatically impairs L-AChR expression. We demonstrate that 3 ␣-and 2 non-␣-subunits assemble into the same receptor. Pharmacological analysis reveals that the prototypical cholinergic agonist nicotine is unable to activate L-AChRs but rather acts as a potent allosteric inhibitor. These results emphasize the role of ancillary proteins for efficient expression of recombinant neurotransmitter receptors and open the way for in vitro screening of novel anthelminthic agents.anthelminthic drug ͉ recombinant receptor expression

There is a medical need for an agent with the positive effects of estrogen on bone and the cardiovascular system, but without the negative effects on reproductive tissue. Raloxifene (LY139481 HCI) is a benzothiophene derivative that binds to the estrogen receptor and inhibits the effects of estrogen on the uterus. In an ovariectomized (OVX) rat model we investigated the effects of raloxifene on bone loss (induced by estrogen deficiency), serum lipids, and uterine tissue. After oral administration of raloxifene for 5 wk (0.1-10 mg/kg per d) to OVX rats, bone mineral density in the distal femur and proximal tibia was significantly greater than that observed in OVX controls (ED50 of 0.03-0.3 mg/kg). Serum cholesterol was lower in the raloxifene-treated animals, which had a minimal effective dose of 0.1 mg/kg and an approximate oral ED50 of 0.2 mg/kg. The effects of raloxifene on bone and serum cholesterol were comparable to those of a 0.1-mg/kg per d oral dose of ethynyl estradiol. Raloxifene diverged dramatically from estrogen in its lack of significant estrogenic effects on uterine tissue. Ethynyl estradiol produced a marked elevation in a number of uterine histologic parameters (e.g., epithelial cell height, stromal eosinophilia). These data suggest that raloxifene has promise as an agent with beneficial bone and cardiovascular effects in the absence of significant uterine effects. (J. Clin. Invest. 1994. 93:63-69.)

A clonal hamster beta cell line (HIT) was established by simian virus 40 transformation of Syrian hamster pancreatic islet cells. Cytoplasmic insulin was detected in all cells by indirect fluorescent antibody staining, and membrane-bound secretory granules were observed ultrastructurally. Acidified-ethanol extracts of HIT cell cultures contained hamster insulin as determined by radioimmunoassay, radioreceptor assay, and bioassay. One subclone at passage 39 contained 2.6 ,ug of insulin per mg of cell protein.[3H]Leucine-labeled HIT insulin and proinsulin were identical to islet-derived proteins when compared by NaDodSO4 polyacrylamide gel electrophoresis of immunoprecipitates. HIT cell insulin secretion was stimulated by glucose, glucagon, and 3-isobutyl-l-methylxanthine. Insulin secretion at ogtimal glucose concentration (7.5 mM) was 2.4 milliunits per 10 cells per hr. Somatostatin and dexamethasone markedly inhibited HIT insulin secretion. The HIT cell line represents a unique in vitro system for studying beta cell metabolism and insulin biosynthesis.Much of the current understanding of insulin biosynthesis and beta cell metabolism has been derived from in vitro studies utilizing isolated islets, either intact or dissociated in monolayer culture. Such studies have been limited by (i) the difficulty of preparing even small quantities of islets, (ii) cellular and hormonal heterogeneity within islets, and (iii) rapid loss of insulin production in vitro. The recent availability of a highly differentiated rat insulinoma tumor line (1) has provided experimental material for biochemical studies including purification and characterization ofpreproinsulin mRNA (2) and cloning and sequence analysis ofcDNA (3). Further in vitro studies would be facilitated by the availability of permanent cell lines possessing functions characteristic of differentiated beta cells. Attempts to establish stable insulin-producing cell lines from primary islet monolayer cultures (4-6), insulinomas (7,8), and hybrids ofislet cells and continuous cell lines (9) have met with only limited success. Simian virus 40 (SV40) transformation of rat islets has yielded continuous cell lines that produced a 30,000-dalton protein antigenically related to insulin (10). Rae et al. (11) have recently derived continuous cell lines from the poorly differentiated Kirkman hamster insulinoma which did not make insulin in vitro but resumed insulin synthesis in vivo.In this communication we describe a continuous beta cell line, established from SV40-transformed Syrian hamster islet cells, that produces substantial quantities of insulin and proinsulin in vitro. Preliminary studies characterizing the insulin product and comparing stimulus-secretion coupling between this transformed hamster beta cell line (HIT) and primary islet cultures are also described.MATERIALS AND METHODS Isolation of Pancreatic Islets. Pancreata were dissociated by the collagenase-digestion technique of Lacy and Kostianovsky (12). DNase I (Worthington; code DP, 23 units/ml) was added ...

The effect of 17i-estradiol on interleukin-6 (IL-6) synthesis was examined in murine bone marrow-derived stromal cell lines, normal human bone-derived cells, and nontransformed osteoblast cell lines from mice and rats. In all these cell types IL-6 production was stimulated as much as 10,000-fold in response to the combination of recombinant interleukin-l (IL-i) and tumor necrosis factor a (TNFa). Addition of 17ft-estradiol in the cultures exerted a dose-dependent inhibition of IL-1-, TNF-, and IL-1 + TNF-induced production of bioassayable IL-6. Testosterone and progesterone (but not 17a-estradiol) also inhibited IL-6, but their effective concentrations were two orders of magnitude higher than 17#-estradiol. 17,#-estradiol also decreased the levels of the IL-6 mRNA. In addition, estradiol inhibited both TNF-induced IL-6 production and osteoclast development in primary bone cell cultures derived from neonatal murine calvaria. The TNF-stimulated osteoclast development was also suppressed by a neutralizing monoclonal anti-IL-6 antibody. This in vitro evidence suggests, for the first time, a mechanistic paradigm by which estrogens might exert at least part of their antiresorptive influence on the skeleton. (J. Clin. Invest. 1992. 89:883-891.)