Osteoclast formation, survival and morphology are highly dependent on exogenous cholesterol/lipoproteins.

Osteoporosis is associated with both atherosclerosis and vascular calcification. No mechanism yet explains the parallel progression of these diseases. Here, we demonstrate that osteoclasts (OCL) depend on lipoproteins to modulate cellular cholesterol levels and that this controls OCL formation and survival. Removal of cholesterol in OCL via high-density lipoprotein or cyclodextrin treatment dose-dependently induced apoptosis, with actin disruption, nuclear condensation and caspase-3 activation. One mechanism linked to the induction of OCL apoptosis was the cell-type-specific failure to induce HMG-CoA reductase mRNA expression, suggesting an absence of feedback regulation of de novo cholesterol biosynthesis. Furthermore, cyclodextrin treatment substantially suppressed essential M-CSF and RANKL-induced survival signaling pathways via Akt, mTOR and S6K. Consistent with these findings, cholesterol delivery via low-density lipoprotein (LDL) significantly increased OCL viability. Interestingly, OCLs from the LDL receptor (LDLR)-/- mouse exhibited reduced size and lifespan in vitro. Remarkably, LDLR+/+ OCL in lipoprotein-deficient medium phenocopied LDLR-/- OCL, while fusion and spreading of LDLR-/- OCL was rescued when cholesterol was chemically delivered during differentiation. With hyperlipidemia being associated with disease of the vascular system and bone, these findings provide novel insights into the selective lipoprotein and cholesterol dependency of the bone resorbing cell. Cell Death and Differentiation (2004) 11, S108-S118. doi:10.1038/sj.cdd.4401399 Published online 12 March 2004

The organization of adherens junctions in mouse osteoblast-like cells (MC3T3-E1) and their modulation by triiodothyronine and 1,25-dihydroxyvitamin D3.

Cadherin-mediated cell-cell adhesion is essential for the development and survival of multicellular tissues. Thus it is hypothesized that these molecules also play a fundamental role for the development and maintenance of bone by mediating cellular crosstalk between osteogenic cells and by providing targets for the sorting and migration of osteogenic precursors toward the bone surface. We describe the localization of cadherin-11 and N-cadherin along the cell margins of mouse osteoblast-like cells, the colocalization of "pancadherin" with alpha-catenin, beta-catenin, p120, and vinculin, and the association of these complexes with the actin microfilaments. Furthermore, we measured the influence of cell confluency and the effects of the osteogenic hormones triiodothyronine (T3) and 1,25-dihydroxyvitamin D3 (D3) on these parameters. By mRNA studies we found the abundantly expressed cadherin-11 being unaffected during T3- and D3-induced osteoblastic differentiation. However, protein levels of N-cadherin and "pancadherin" were strongly suppressed by D3. We also observed a clear distinction in cadherin immunolocalization when comparing confluent control and confluent hormone-treated cultures. Immunoprecipitation experiments indicated that vinculin is part of the junctional complex, and that the association of "pancadherin"/beta-catenin is strongly increased after treatment with T3 which might influence the functional competence of cell-cell contacts. Thus, this study demonstrates the molecular organization of adherens junctions in mouse osteoblastic MC3T3-E1 cells and their sensitivity to the osteogenic factors T3 and D3 in confluent cultures.

Tri-iodothyronine inhibits multilayer formation of the osteoblastic cell line, MC3T3-E1, by promoting apoptosis.

Cell death through apoptosis is a well-known mechanism for maintaining homoeostasis in many developmental and pathological processes. We have recently presented evidence for the occurrence of apoptosis during the formation of bone-like tissue in vitro. MC3T3-E1 osteoblast-like cells in culture develop features of the osteoblastic phenotype and form many cell layers embedded in extracellular matrix which can mineralise. Tri-iodothyronine (T3), even though it enhances the expression of many osteoblastic features, attenuates the multilayer formation to about two layers. The aim of this study was to investigate how T3 prevents multilayer formation. MC3T3-E1 cells were seeded at different densities and cultured for up to 2 weeks. Thereafter we analysed proliferation rate and the distribution of the phases of the cell cycle and studied apoptosis. We found that T3 did not inhibit DNA synthesis. Analysis of the cell cycle phases showed an increase in the number of cells in G0/G1 with increasing cell density, but no significant effect of T3 treatment was found. Morphological investigations showed apoptotic features in both cell layers and culture supernatants. The cells exhibited typical plasma membrane blebbings, chromatin condensation, DNA fragmentation and phagocytosed apoptotic bodies. T3 treatment significantly increased the number of apoptotic cells. We conclude from our data that T3 inhibits multilayer formation of MC3T3-E1 cells by increasing the rate of apoptosis and not by inhibition of proliferation. Because apoptosis is a fundamental regulatory event during bone tissue differentiation, our findings emphasise the importance of thyroid hormones in bone maintenance and development.

1,25-Dihydroxy vitamin D3 and tri-iodothyronine stimulate the expression of a protein immunologically related to osteocalcin.

Osteocalcin (OC), a bone-specific protein, is a marker of late osteoblastic differentiation. Its expression is influenced by various growth factors and hormones. We investigated the effect of 1, 25-dihydroxy vitamin D3 (D3) and tri-iodothyronine (T3) on OC expression in osteoblast-like MC3T3-E1 cells. A heterologous OC green fluorescence protein (GFP) fusion vector was established and expressed to study possible effects on protein transport. Immunostaining of endogenous OC revealed a significant increase in the percentage of positive cells after D3 and T3 treatment. This was consistent for MC3T3-E1 cells as well as nonosteogenic NIH-3T3 and mammary carcinoma cells, but not for neuroblastoma cells. The perinuclear immunostaining corresponded to the NBD C6 ceramide Golgi staining. Conversely, we found a strong induction of OC in MC3T3-E1 cells at the mRNA and protein levels only with T3 and not with D3. OC mRNA and protein expression was not detected in NIH fibroblasts. OC GFP transfection experiments indicate rapid transport and secretion of OC, because OC GFP was not found to be accumulated at intracellular compartments after hormone treatment. We conclude that the strong perinuclear immunostaining does not represent OC but a protein immunologically related to OC, as indicated by preabsorption experiments. The expression of this OC epitope-sharing protein is regulated by both D3 and T3 in the osteoblastic MC3T3-E1 and in nonosteogenic cells.

Characterization of the mouse insulin-like growth factor binding protein 4 gene regulatory region and expression studies.

Insulin-like growth factor binding protein 4 (IGFBP-4) is known as a potent inhibitor of IGFs action in various cell types. In this study, the mouse IGFBP-4 gene 5' flanking region, the IGFBP-4 mRNA expression, and the IGFBP-4s intracellular transport were investigated. The regulatory region exhibits all elements typical for an eukaryotic TATA element containing promoter and was found to also contain functional elements to direct transcriptional activation of a luc reporter gene construct that gradually decreased by 5' unidirectional deletions. Responsiveness of the IGFBP-4 promoter activity was tested with thyroid hormone and found only within extended constructs but not when a potential TRalpha1-binding site had been deleted. By using exon specific probes, we observed a varying expression pattern of IGFBP-4 transcripts in three rodent cell lines. Surprisingly, mouse fibroblastic NIH/3T3 cells displayed exclusively about a 2.0-kb transcript apparently lacking the IGFBP-4 mRNA 5' region. Studies on the intracellular transport by establishment of an IGFBP4/green fluorescent protein (GFP) fusion protein clearly demonstrate that IGFBP-4 is transported continuously along the intracellular secretory pathway and is excluded from other intracellular compartments. The description of the genomic IGFBP-4 region in the mouse now opens new perspectives for further clarification of the role of IGFBP-4 in growth and development.

Triiodothyronine, a regulator of osteoblastic differentiation: depression of histone H4, attenuation of c-fos/c-jun, and induction of osteocalcin expression.

Thyroid hormones influence growth and differentiation of bone cells. In vivo and in vitro data indicate their importance for development and maintenance of the skeleton. Triiodothyronine (T3) inhibits proliferation and accelerates differentiation of osteoblasts. We studied the regulatory effect of T3 on markers of proliferation as well as on specific markers of the osteoblastic phenotype in cultured MC3T3-E1 cells at different time points. In parallel to the inhibitory effect on proliferation, T3 down-regulated histone H4 mRNA expression. Early genes (c-fos/c-jun) are highly expressed in proliferating cells and are down-regulated when the cells switch to differentiation. When MC3T3-E1 cells are cultured under serum-free conditions, basal c-fos/c-jun expressions are nearly undetectable. Under these conditions, c-fos/c-jun mRNAs can be stimulated by EGF, the effect of which is attenuated to about 46% by T3. In addition, T3 stimulated the expression at the mRNA and protein level of osteocalcin, a marker of mature osteoblasts and alkaline phosphatase activity. All these effects were more pronounced when cells were cultured for more than 6 days. These data indicate that T3 acts as a differentiation factor in osteoblasts by influencing the expression of cell cycle-regulated, of cell growth-regulated, and of phenotypic genes.

Effects of triiodothyronine on the morphology of cells and matrix, the localization of alkaline phosphatase, and the frequency of apoptosis in long-term cultures of MC3T3-E1 cells.

The effects of triiodothyronine (T3) on the localization and morphology of alkaline phosphatase (ALP)-positive cells, matrix formation, and apoptosis in MC3T3-E1 cells cultured up to 6 weeks were investigated by light and electron microscopy. Cell size, shape, and frequency of apoptosis were measured histomorphometrically. At all time points both ALP-positive and -negative cells were observed histochemically. Control cultures older than 3 weeks were characterized by colonies of small cuboidal ALP-positive cells. Cross sections revealed that these areas corresponded to unmineralized nodules. The thickening was caused by local accumulation of extracellular matrix. The internodular regions were characterized by ALP-positive spindle-shaped cells randomly distributed throughout all cell layers. Apoptotic nuclei were found within a frequency of 0.2%-1%. With increasing culture time the percentage of apoptotic cells became higher in the nodules. T3 treatment inhibited cell proliferation and stimulated ALP activity. After confluence, T3-treated cultures reached two to three cell layers at maximum and showed a different morphology and histochemical staining pattern. ALP-positive cells were stellar shaped and larger than unstained cells. Small ALP-positive colonies suggested nodule formation; however, the most striking differences between T3-treated and control cultures were a decrease in the amount of extracellular matrix with only few collagen fibers and the absence of local matrix accumulation. Furthermore, the number of apoptotic nuclei was increased. Our data extend beyond previous observations on the role of thyroid hormones in osteoblastic differentiation. Besides their effects on proliferation and cell morphology, they influence ALP activity, matrix composition, nodule formation, and apoptotic transformation.

Effects of triiodothyronine on morphology, growth behavior, and the actin cytoskeleton in mouse osteoblastic cells (MC3T3-E1).

We investigated the effects of thyroid hormone treatment on morphology, growth behaviour, and cytoskeletal structures of long-term cultured MC3T3-E1 cells. Morphological investigations were carried out on native cells by phase contrast microscopy and on epon-embedded semithin sections. The area covered by the cell and matrix layers (tissue-like area), percent extracellular matrix, average height of tissue-like area, and length and height of single cells were measured histomorphometrically on the cross sections. F-actin was analyzed histochemically and quantitated after fluorochrome-labeled phalloidin staining using confocal microscopy and fluorometry. Significant differences between control and T3-treated cells were found after confluency, but not in subconfluent cultures. Control cells continued to proliferate forming multilayers, and produced increasing amounts of extracellular matrix. In contrast, T3-treated cells stopped to proliferate forming two cell layers at the maximum. These cells were flattened, distinctly enlarged, and polygonal in shape. Histochemical staining for F-actin revealed three different staining patterns, depending on the position of the cell within the multilayer of control cultures. Basal cells contained a large number of thick stress fibers in parallel arrangement. Intermediate cells exhibited only a few thick actin filament bundles located at the outermost periphery. The superficial cells were characterized by a large number of thin, parallel-oriented microfilament bundles extending across the entire cytoplasm. The actin pattern of T3-treated cells resembled that of the basal cell layer of the control cells. The amount of F-actin increased with the prolonged T3 treatment. We conclude from these data that the known specific cellular responses to T3 treatment are accompanied by significant morphological alterations indicating pivotal effects of thyroid hormones on osteoblastic differentiation.