Interaction of calcitonin gene-related peptide (CGRP) with its receptors leads to stimulation of adenylyl cyclase and/or phospholipase C (PLC). While regulation of adenylyl cyclase is thought to involve the G-protein Gs, it is not known whether activation of PLC results from coupling the receptor to Gq family proteins or whether βγ subunits released from receptor-activated Gs activate PLC. We used human bone cells OHS-4 bearing CGRP receptors in which CGRP activates only the PLC signaling pathway to determine how CGRP acts. CGRP increased the concentration of intracellular calcium ([Ca2+]i) within 5 s via a Ca2+ influx through voltage-gated calcium channels and by mobilizing calcium from the endoplasmic reticulum. The activation of effectors, like PLC coupled to G-proteins, is the early event in the pathway leading to inositol 1,4,5-trisphosphate formation, which is responsible for Ca2+ mobilization. Western blotting demonstrated a range of PLC-β isoforms (β1, β3, β4, but not β2) and G-proteins (Gαq/11 and Gαs). Only phospholipase C-β1 is involved in the mobilization of Ca2+ from the endoplasmic reticulum of Fura-2-loaded confluent OHS-4 cells and the formation of inositol 1,4,5-trisphosphate by CGRP; PLC-γ have no effect. Activation of PLC-β1 by CGRP involves the Gαq/11 subunit, which is insensitive to pertussis toxin, but not Gβγ subunits. We therefore believe that CGRP causes the activation of two separate G-proteins.
Abstract Skeletal unloading induced by hindlimb suspension in rats reduces bone formation and induces osteopenia, but its effect on adipogenesis is unknown. We assessed the effects of unloading and transforming growth factor (TGF) β2 on bone marrow stromal cell adipocyte differentiation in relation with osteoblast differentiation. Skeletal unloading rapidly (4-7 days) decreased osteoblast transcription factor Runx2, osteocalcin (OC), and type I collagen messenger RNA (mRNA) levels and reduced bone formation in the long bone metaphysis. Conversely, unloading increased expression of the adipocyte transcription factor peroxisome proliferator-activated receptor γ2 (PPARγ2) at 4 days and increased expression of the adipocyte differentiation genes lipoprotein lipase (LPL) and aP2 in the bone marrow stroma at 7 days. Consistently, unloading increased the number and volume of adipocytes in the bone marrow stroma. Continuous (0-7 days) and late (4-7 days) treatments with TGF-β2 corrected the abnormal expression of Cbfa1/Runx2, OC, and type I collagen mRNAs and normalized bone formation in unloaded metaphyseal bone. Moreover, both TGF-β2 treatments decreased PPARγ2 and C/EBPα mRNA levels at 4 days and normalized aP2 and LPL expression and adipocyte number and volume at 7 days. These results show that skeletal unloading increases adipocyte differentiation concomitantly with inhibition of osteoblast differentiation. These abnormalities are prevented and reversed by TGF-β2, suggesting a role for TGF-β in the control of adipogenic differentiation in the bone marrow stroma.
mRNAs were isolated from 2 patients suffering from a familial form of a rare variant of medullary carcinoma of the thyroid (MTC), called mixed follicular and medullary carcinoma. The presence of calcitonin (CT) and thyroglobulin (Tg) mRNAs was checked by northern and in situ hybridization and compared with immunohistochemical results. In each case, mRNAs hybridizing to probes specific for CT and Tg were detected. Both proteins were quantified by radioimmunoassay determination in tissue extracts. Patient 1 had 20 ng Tg and 68 ng CT per micrograms total protein, and patient 2 had 0.4 ng Tg and 1.7 ng CT per micrograms total protein. Northern analysis showed that mixed carcinoma expressed several species of both CT mRNAs and Tg mRNAs. The main Tg transcripts present in neoplastic cells (8.5 and 4.8 kb for patient 1 and patient 2) were identical to or smaller than those of normal thyroid tissue (8.5 kb). The tumor CT mRNA (1 kb) was identical to that of normal tissue. In situ hybridization confirmed the presence of CT and Tg mRNA in the great majority of tumor cells. Furthermore, the presence of small amounts of organified iodine was evidenced by analytical ion microscopy in 35% of these cells. This raises an important question regarding the histogenesis of this tumor.
Bone morphogenetic protein-2 (BMP-2) stimulates the differentiation of osteoblastic cells. However, the mechanisms involved in this effect are not well characterized. In this study, we determined the role of the cell-cell adhesion molecules N-cadherin and E-cadherin in the promotion of osteoblast differentiation by BMP-2 in immortalized human neonatal calvaria (IHNC) cells. In cells cultured in aggregates, recombinant human BMP-2 (rhBMP-2) increased messenger RNA levels for alkaline phosphatase (ALP), the osteoblast specific transcription factor Osf2/Cbfa1 and osteocalcin, and enhanced in vitro osteogenesis in long-term culture. RT-PCR, immunocytochemical, and Western blot analyses showed that IHNC cells express E-cadherin, N-cadherin, and neural cell adhesion molecule (N-CAM) mRNA and protein. Treatment with rhBMP-2 induced a rapid and transient increase in N-cadherin and E-cadherin but not N-CAM, mRNA, and protein levels. Incubation with the RNA polymerase II inhibitor 5, 6-dichloro-1-beta-D-ribofuranosyl benzimidazole prevented the upregulation of N- and E-cadherins induced by rhBMP-2, suggesting that transcription is necessary for this effect. N- and E-cadherins were functional because rhBMP-2 increased cell-cell adhesion in a cell aggregation assay, and this effect was largely blocked by N-cadherin- and E-cadherin-neutralizing antibodies. In addition, N- and E-cadherin antibodies decreased the basal ALP activity and completely suppressed the rhBMP-2-induced increase in ALP activity and mRNA levels. Furthermore, anti-N-cadherin or anti-E-cadherin antibodies markedly decreased Osf2/Cbfa1 mRNA levels and abolished the rhBMP-2-induced increased Osf2/Cbfa1 expression, and reduced the increased osteocalcin mRNA levels induced by rhBMP-2. We conclude that rhBMP-2 rapidly and transiently increases N- and E-cadherin expression, and this effect mediates the rhBMP-2-induced early promotion of cell-cell adhesion and osteoblast marker gene expression in human calvaria cells.
La calcitonine, hormone-hypocalcemiante hypophos-phatemiante, est aussi le marqueur specifique d'une tumeur humaine. Les sequences de cette hormone divergent entre les vertebres mammaliens et non mammaliens. La presence chez l'homme de molecules reagissant avec des anticorps anti calcitonine de saumon pose probleme de l'existence d'un deuxieme gene Le but de ce travail a ete de determiner, pour la premiere fois, la structure de l'ARNm de la calcitonine d'un vertebre non mammalien, La calcitonine de poulet a ete choisie car cette molecule est antigeniquement apparentee a celle de saumon. Un dosage radio immunologique heterologue, specifique de la calcitonine aviaire, a ete realise et les cellules ultimobranchiales productrices de l'hormone localisees. Les ARNm des glandes ultimobranchiales ont ete extraits et le messager de la calcitonine identifie par traduction et immunoprecipitation specifique. L'ARNm de la calcitonine a ete purifie et son ADNc clone dans pBR322. Un clone selectionne par la technique d'hybridation selection positive a permis d'etablir la sequence presque complete du messager de la calcitonine. Utilisant ce dernier ADN comme sonde, une banque genomique de poulet a ete criblee; un phage contenant le gene de la calcitonine a ete isole, et sa sequence partiellement determinee. La sequence complete du precurseur de la calcitonine aviaire a ainsi ete elucidee. Cette polyproteine a la meme organisation que celle des precurseurs humains et murins : deux petites peptides encadrant la molecule de calcitonine a ses extremites N et C terminales, et les sites de clivage proteolytique et d'amidation. La comparaison des sequences des ARNm des calcitonines aviaire et mammaliennes montre que la sequence de la molecule de la calcitonine ainsi que celle de la sequence signal sont fortement conservees, par contre les sequences des petides N et C terminaux ont varie de facon lineaire pendant l'evolution. Une sonde specifique du messager de poulet, a demontre qu'un gene, codant pour une calcitonine de type vertebre non mammalien, etait exprime dans des tumeurs thyroidiennes humaine et murine.
