Deletion of osteopontin or bone sialoprotein induces opposite bone responses to mechanical stimulation in mice.

Osteopontin (OPN) and Bone Sialoprotein (BSP) are co-expressed in bone and display overlapping and complementary physiological properties. Both genes show a rapid expression response to mechanical stimulation. We used mice with single and double deletions (DKO) of BSP and OPN to assess the specificity of their roles in skeletal adaptation to loading. Two-month-old Wild-Type (WT), BSP knockout (BSP-/-), OPN-/- and DKO male mice were submitted to two mechanical stimulation regimen (n = 10 mice/group) respectively impacting trabecular bone (Hypergravity, HG) and cortical bone (Whole Body Vibration, WBV). HG increased trabecular bone volume (BV/TV) in WT femur through reduced resorption, and in BSP-/- mice femur and vertebra through increased bone formation. In contrast, HG increased the turnover of OPN-/- bone, resulting in reduced femur and vertebra BV/TV. HG did not affect DKO bones. Similarly, WBV increased cortical thickness in BSP-/- mice and decreased it in OPN-/-, without affecting structurally WT and DKO bone. Vibrated BSP-/- mice displayed increased endocortical bone formation with a drop in Sclerostin expression, and reduced periosteal osteoclasts with lower Rankl and Cathepsin K expression. In contrast, vibrated OPN-/- endocortical bone displayed decreased formation and increased osteoclast coverage. Therefore, under two regimen (HG and WBV) targeting distinct bone compartments, absence of OPN resulted in bone loss while lack of BSP induced bone gain, reflecting divergent structural adaptations. Strikingly, absence of both proteins led to a relative insensitivity to either mechanical challenge. Interplay between OPN and BSP thus appears as a key element of skeletal response to mechanical stimulation.

Deletion of OPN in BSP knockout mice does not correct bone hypomineralization but results in high bone turnover.

The two SIBLING (Small Integrin Binding Ligand N-linked Glycoproteins), bone sialoprotein (BSP) and osteopontin (OPN) are expressed in osteoblasts and osteoclasts. In mature BSP knockout (KO, -/-) mice, both bone formation and resorption as well as mineralization are impaired. OPN-/- mice display impaired resorption, and OPN is described as an inhibitor of mineralization. However, OPN is overexpressed in BSP-/- mice, complicating the understanding of their phenotype. We have generated and characterized mice with a double KO (DKO) of OPN and BSP, to try and unravel their respective contributions. Despite the absence of OPN, DKO bones are still hypomineralized. The SIBLING, matrix extracellular phosphoglycoprotein with ASARM motif (MEPE) is highly overexpressed in both BSP-/- and DKO and may impair mineralization through liberation of its ASARM (Acidic Serine-Aspartate Rich MEPE associated) peptides. DKO mice also display evidence of active formation of trabecular, secondary bone as well as primary bone in the marrow-ablation repair model. A higher number of osteoclasts form in DKO marrow cultures, with higher resorption activity, and DKO long bones display a localized and conspicuous cortical macroporosity. High bone formation and resorption parameters, and high cortical porosity in DKO mice suggest an active bone modeling/remodeling, in the absence of two key regulators of bone cell performance. This first double KO of SIBLING proteins thus results in a singular, non-trivial phenotype leading to reconsider the interpretation of each single KO, concerning in particular matrix mineralization and the regulation of bone cell activity.

Validation of an in vitro 3D bone culture model with perfused and mechanically stressed ceramic scaffold.

An engineered three dimensional (3D) in vitro cell culture system was designed with the goal of inducing and controlling in vitro osteogenesis in a reproducible manner under conditions more similar to the in vivo bone microenvironment than traditional two-dimensional (2D) models. This bioreactor allows efficient mechanical loading and perfusion of an original cubic calcium phosphate bioceramic of highly controlled composition and structure. This bioceramic comprises an internal portion containing homogeneously interconnected macropores surrounded by a dense layer, which minimises fluid flow bypass around the scaffold. This dense and flat layer permits the application of a homogeneous loading on the bioceramic while also enhancing its mechanical strength. Numerical modelling of constraints shows that the system provides direct mechanical stimulation of cells within the scaffold. Experimental results establish that under perfusion at a steady flow of 2 µL/min, corresponding to 3 ≤ Medium velocity ≤ 23 µm/s, mouse calvarial cells grow and differentiate as osteoblasts in a reproducible manner, and lay down a mineralised matrix. Moreover, cells respond to mechanical loading by increasing C-fos expression, which demonstrates the effective mechanical stimulation of the culture within the scaffold. In summary, we provide a "proof-of-concept" for osteoblastic cell culture in a controlled 3D culture system under perfusion and mechanical loading. This model will be a tool to analyse bone cell functions in vivo, and will provide a bench testing system for the clinical assessment of bioactive bone-targeting molecules under load.

New insight into the bony labyrinth: a microcomputed tomography study.

OBJECTIVES: To visualize and quantify the morphology and mineralization of the developing fetal human bony labyrinth, using 3D-microcomputed tomography (3D-microCT) imaging. METHODS: Eleven right temporal bones from late second and third trimester fetuses were used in this prospective pilot study. After fixation in 10% formalin solution, all samples underwent a microcomputed tomography (microCT) scan, permitting the 3D imaging of the bony labyrinth as well as the quantitative assessment of mineral density, angular distances and dimensions of inner ear components the progression of ossification was precised with histological observations. RESULTS: Our findings show different rates of growth among the semicircular canals, the vestibular aqueduct, the oval window, the round window and the cochlea. The final sizes of the cochlea and round window are achieved at 23 weeks of gestation, with heights of 5mm and 2mm, respectively. The oval window reaches adult size at 35 weeks, whereas the vestibular aqueduct will attain adult size after birth. An increasing degree of torsion of each semicircular canal is observed during fetal development. The superior semicircular canal achieves adult size at 24 weeks, before the posterior and the lateral canals (25 weeks). The time-course of ossification and mineralization observed in structures and confirmed by histology. CONCLUSIONS: During this developmental period poorly studied until now, our findings suggest that each part of the bony labyrinth follows distinct growth and ossification kinetics trajectories, some of these reaching their adult size only after birth.

Cellular accumulation and distribution of uranium and lead in osteoblastic cells as a function of their speciation.

Uranium (U) and lead (Pb) are accumulated and fixed for long periods in bone, impairing remodeling processes. Their toxicity to osteoblasts, the cells responsible for bone formation, is poorly documented. It has been previously shown that cytotoxicity and phenotypic effects of both metals on osteoblasts are highly influenced by metal speciation. Differences in sensitivity between cell types have been underlined as well. In this paper, cellular accumulation of U and Pb in cultured and primary osteoblastic cells was assessed by trace element analysis. Distribution of different species at the cell scale was investigated by electron microscopy. Internalization of both metals was shown to be correlated to cytotoxicity and population growth recovery after exposure. For each metal, the amount of metal uptake leading to 50% cell death was shown to be speciation-dependent. Scanning and transmission electron microscopy showed the formation of precipitates with phosphate in lysosomes for both metals, whose role in toxicity or cell defence remains to be clarified. Although a clear link was established between cytotoxicity and accumulation, differences in sensitivity observed in terms of speciation could not be fully explained and other studies seem necessary.

Low dose beta-blocker prevents ovariectomy-induced bone loss in rats without affecting heart functions.

Findings from animal studies have suggested that bone remodeling is under beta-adrenergic control. However, the level of adrenergic inhibition required to achieve the most favorable effects on the skeleton remains unknown. To address this question, we compared the effects of low (0.1 mg/Kg/day), medium (5 mg/Kg/day) or high (20 mg/Kg/day) doses of propranolol given 5 days per week for 10 weeks in ovariectomized (OVX) rats. Characteristics of bone microarchitecture, biomechanical properties and bone turnover were investigated, whilst heart functions were assessed by echocardiography and catheterization of the left ventricle. We first confirmed the expression of Adrbeta2R and the absence of Adrbeta1R on osteoblasts by PCR and confocal microscopy. We then showed that low dose propranolol prevented OVX induced bone loss by increasing bone formation (+30% of MAR vs. placebo, P = 0.01) and decreasing bone resorption (-52% of osteoclast surface on bone surface vs. placebo, P = 0.01). Consequently, rats receiving 0.1 mg/kg/day propranolol displayed higher stress (+27%), intrinsic energy (+28.7%) and Young's Modulus in compression versus placebo (all, P < 0.05). No significant effects on heart hemodynamic parameters were found in rats receiving this dose. In contrast, medium and high doses of propranolol had a negative effect on heart functions but no significant protective effects on bone mass in ovariectomized rats. These results, consistent with the dominant nature of the high bone mass phenotype and normal heart function of Adrbeta2R-deficient mice, suggest that low doses of beta-blockers may have a therapeutic utility in the treatment of osteoporosis with high selectivity for bone tissues.

Cytotoxic and phenotypic effects of uranium and lead on osteoblastic cells are highly dependent on metal speciation.

Bone is one of the main retention organs for uranium (U) and lead (Pb). The clinical effects of U or Pb poisoning are well known: acute and chronic intoxications impair bone formation. However, only few studies dealt with the cellular and molecular mechanisms of their toxicity. The purpose of this study was to investigate acute cytotoxicity of U and Pb and their phenotypic effects on rat and human osteoblasts, the cells responsible for bone formation. The most likely species of the toxicants in contact with cells after blood contamination were selected for cell exposure. Results showed that the cytotoxic effect of U and Pb is highly dependent on their speciation. Thus, Pb was cytotoxic when left free in the exposure medium or when complexed with carbonate, cysteine or citrate, but not when complexed with albumin or phosphate, under an insoluble form. U was cytotoxic whatever its speciation, but differences in sensitivity were observed as a function of speciation. Population growth recovery could be obtained after exposure to low doses of U or Pb, except for some U-carbonate complexes which had irreversible effects whatever the dose. The activation of two markers of bone formation and mineralization, osteocalcin and bone sialoprotein (BSP), was observed after exposure to non-toxic doses or non-toxic species of U or Pb while their inhibition was observed after toxic exposure to both metals. This work provides new elements to better understand the complex mechanisms of U and Pb toxicity to osteoblasts. Our results also illustrate the importance of a strictly controlled speciation of the metals in toxicological studies.

Expression of Semaphorin-3A and its receptors in endochondral ossification: potential role in skeletal development and innervation.

Bone tissue is densely innervated, and there is increasing evidence for a neural control of bone metabolism. Semaphorin-3A is a very important regulator of neuronal targeting in the peripheral nervous system as well as in angiogenesis, and knockout of the Semaphorin-3A gene induces abnormal bone and cartilage development. We analyzed the spatial and temporal expression patterns of Semaphorin-3A signaling molecules during endochondral ossification, in parallel with the establishment of innervation. We show that osteoblasts and chondrocytes differentiated in vitro express most members of the Semaphorin-3A signaling system (Semaphorin-3A, Neuropilin-1, and Plexins-A1 and -A2). In vitro, osteoclasts express most receptor chains but not the ligand. In situ, these molecules are all expressed in the periosteum and by resting, prehypertrophic and hypertrophic chondrocytes in ossification centers before the onset of neurovascular invasion. They are detected later in osteoblasts and also osteoclasts, with differences in intensity and regional distribution. Semaphorin-3A and Neuropilin-1 are also expressed in the bone marrow. Plexin-A3 is not expressed by bone cell lineages in vitro. It is detected early in the periosteum and hypertrophic chondrocytes. After the onset of ossification, this chain is restricted to a network of cell processes in close vicinity to the cells lining the trabeculae, similar to the pattern observed for neural markers at the same stages. After birth, while the density of innervation decreases, Plexin-A3 is strongly expressed by blood vessels on the ossification front. In conclusion, Semaphorin-3A signaling is present in bone and seems to precede or coincide at the temporal but also spatial level with the invasion of bone by blood vessels and nerve fibers. Expression patterns suggest Plexin-A3/Neuropilin-1 as a candidate receptor in target cells for the regulation of bone innervation by Semaphorin-3A.

Expression of leukemia inhibitory factor (LIF)/interleukin-6 family cytokines and receptors during in vitro osteogenesis: differential regulation by dexamethasone and LIF.

The leukemia inhibitory factor/interleukin-6 (LIF/IL-6) family of cytokines is known to play a major role in bone physiology. Although much work has focused on the regulation of bone resorption by IL-6 and related cytokines, their effects on osteoblast development and bone formation have not been as well studied. Previously, we reported that LIF inhibits, in a non-IL-6-dependent manner, osteoblast differentiation and bone nodule formation in the rat calvaria (RC) model, an effect that is antagonized by dexamethasone (Dex). The culture time-sensitive window suggested that LIF targets late preosteoblasts or early osteoblasts, and that this stage-specific effect coincided with a period of low endogenous production of LIF and IL-6. To detect potential crosstalk between members of this family, we have extended these observations by assessing the expression levels of other LIF/IL-6 cytokines (CNTF, OSM, IL-11, CT-1) and their receptors in the same RC cell model treated with or without LIF or Dex. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that IL-11 and its receptor, CNTF and its receptor, LIFR, and gp130 were constitutively expressed throughout the culture period. Expression of CT-1 and OSM increased with culture time - that is, with osteoblast differentiation - whereas the specific receptor for OSM (OSMR) was highly expressed at early timepoints and either plateaued or decreased thereafter. Continuous treatment with Dex (10(-8) mol/L) inhibited the endogenous production of IL-6, LIF, OSM, IL-11R, and OSMR, but had no detectable effect on the expression of IL-11, CT-1, CNTF, CNTFR, LIFR, or gp130. Finally, treatment with exogenously added LIF stimulated IL-6, LIF, LIFR, and OSMR, but had no other detectable effects. These data indicate that multiple members of the LIF/IL-6 family and their receptors are expressed in RC cell cultures, and are differentially regulated by Dex and LIF, suggesting that these cytokines play a complex and interdependent role, further modulated by glucocorticoid levels, in osteoprogenitor differentiation and bone nodule formation.

Investigation of osteocalcin, osteonectin, and dentin sialophosphoprotein in developing human teeth.

Biochemical investigations in rodents have shown that numerous mineralized matrix proteins share expression in bone, dentin, and cementum. Little information is available regarding the expression pattern of these proteins in human tissues, particularly during tooth formation. The aim of this study was to identify the expression pattern of the two major noncollagenous proteins of bone and dentin, osteocalcin (OC) and osteonectin (ON), in comparison to the dentin-specific protein, dentin sialophosphoprotein (DSPP). Mandibles from fetuses (5-26 weeks), neonate autopsies, forming teeth from 10-12-year-old patients, third molars extracted for orthodontic reasons, and bone tumors were collected with approval from the National Ethics Committee. Human OC, ON, and DSPP mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR) in fetal mandibles (5-11 weeks) and in primary cell cultures of dental pulp. In addition, OC, ON, and DSPP proteins were localized in forming human mineralized tissues using immunohistochemistry. In vivo, DSPP expression was associated with tooth terminal epithelial-mesenchymal interaction events, amelogenesis and dentinogenesis. Transient DSPP expression was seen in the presecretory ameloblasts with continuous expression in the odontoblasts. In contrast, both osteoblasts and odontoblasts showed a temporal gap between OC and ON expression in early development. ON was expressed in the initial stages of cytodifferentiation, whereas OC was expressed only during the later stages, especially in the teeth. At the maturation stage of enamel formation, both proteins were detected in odontoblasts and their processes within the extracellular matrix. In contrast to bone, OC was not localized extracellularly within the collagen-rich dentin matrix (predentin or intertubular dentin), but was found in the mature enamel. ON was present mostly in the nonmineralized predentin. These results demonstrate for the first time that both OC and ON are produced by human odontoblasts and determine the expression pattern of DSPP in human teeth, and suggest that OC and ON move inside the canalicule via odontoblast cell processes becoming localized to specific extracellular compartments during dentin and enamel formation. These distinct extracellular patterns may be related to the nature of DSPP, OC, and ON interactions with other matrix-specific macromolecules (i.e., amelogenin, dentin matrix protein-1) and/or to the polarized organization of odontoblast secretion as compared with osteoblasts.

Biphasic effects of leukemia inhibitory factor on osteoblastic differentiation.

Leukemia inhibitory factor (LIF) is a cytokine produced by multiple cell types including osteoblasts and which is active on bone metabolism. We have previously shown that in a bone nodule forming in vitro model of osteogenesis, the fetal rat calvaria (RC) cell model, LIF inhibits osteoblast differentiation, acting on late osteoprogenitors and/or early osteoblasts. These results are in contrast to in vivo experiments, in which LIF has been found to increase bone formation. To resolve this discrepancy, we have tested the effect of LIF on rat bone marrow (RBM) stromal cell cultures, an in vitro model encompassing earlier osteoprogenitor stages. LIF inhibited cell growth in early, proliferating RBM cultures, but increased the culture saturation density. The effect of LIF on bone nodule formation in this model was cell density dependent and biphasic. Continuous treatment with LIF reduced the number of bone nodules present in confluent, more mature cultures, and the inhibitory effect was strongest when cells were plated at higher cell density than lower. In contrast, during the early stages of RBM culture, nodule numbers were higher in LIF-treated dishes than in controls, and this effect was greater in lower density cultures. Acute LIF treatment restricted to early time points increased the final number of bone nodules formed in mature RBM cell cultures, but not in RC cell cultures. Our results indicate that LIF exerts complex, stage-specific effects on osteoprogenitor recruitment, differentiation, and bone formation, and that the effects are cell nonautonomous, in the rat bone marrow stromal cell model. J. Cell. Biochem. Suppl. 36: 63-70, 2001.

Genetic analysis reveals different functions for the products of the thyroid hormone receptor alpha locus.

Thyroid hormone receptors are encoded by the TRalpha (NR1A1) and TRbeta (NR1A2) loci. These genes are transcribed into multiple variants whose functions are unclear. Analysis by gene inactivation in mice has provided new insights into the functional complexity of these products. Different strategies designed to modify the TRalpha locus have led to strikingly different phenotypes. In order to analyze the molecular basis for these alterations, we generated mice devoid of all known isoforms produced from the TRalpha locus (TRalpha(0/0)). These mice are viable and exhibit reduced linear growth, bone maturation delay, moderate hypothermia, and reduced thickness of the intestinal mucosa. Compounding TRalpha(0) and TRbeta(-) mutations produces viable TRalpha(0/0)beta(-/-) mice, which display a more severe linear growth reduction and a more profound hypothermia as well as impaired hearing. A striking phenotypic difference is observed between TRalpha(0/0) and the previously described TRalpha(-/-) mice, which retain truncated TRDeltaalpha isoforms arising from a newly described promoter in intron 7. The lethality and severe impairment of the intestinal maturation in TRalpha(-/-) mice are rescued in TRalpha(0/0) animals. We demonstrate that the TRDeltaalpha protein isoforms, which are natural products of the TRalpha locus, are the key determinants of these phenotypical differences. These data reveal the functional importance of the non-T3-binding variants encoded by the TRalpha locus in vertebrate postnatal development and homeostasis.

Kinetics of osteoprogenitor proliferation and osteoblast differentiation in vitro.

Fetal rat calvaria cells plated at very low density generate discrete colonies, some of which are bone colonies (nodules) from individual osteoprogenitors that divide and differentiate. We have analyzed the relationship between cell proliferation and acquisition of tissue-specific differentiation markers in bone colonies followed individually from the original single cell to the fully mineralized state. The size distribution of fully formed nodules is unimodal, suggesting that the coupling between proliferation and differentiation of osteoprogenitor cells is governed by a stochastic element, but distributed around an optimum, corresponding to the peak colony size/division potential. Kinetic analysis of colony growth showed that osteoprogenitors undergo 9-10 population doublings before the appearance of the first morphologically differentiated osteoblasts in the developing colony. Double immunolabeling showed that these proliferating cells express a gradient of bone markers, from proliferative alkaline phosphatase-negative cells at the periphery of colonies, to postmitotic, osteocalcin-producing osteoblasts at the centers. An inverse relationship exists between cell division and expression of osteocalcin, the latter being restricted to late-stage, BrdU-negative osteoblasts, while the expression of all other markers is acquired before the cessation of proliferation, but not concomitantly. Bone sialoprotein expression is biphasic, detectable in some of the early, alkaline phosphatase-negative cells, and again later in both late preosteoblast (BrdU-positive) and osteoblast (BrdU-negative, osteocalcin-positive) cells. In late-stage, heavily mineralized nodules, staining for osteocalcin and bone sialoprotein is not detectable in the oldest/most mature cells. Our observations support the view that the bone nodule "tissue-like" structure, originating from a single osteoprogenitor and finally encompassing mineralized matrix production, recapitulates successive stages of the osteoblast differentiation pathway, in a proliferation/maturation sequence. Understanding the complexity of the proliferation/differentiation kinetics that occurs within bone nodules will aid in the qualitative and/or quantitative interpretation of tissue-specific marker expression during osteoblastic differentiation.

Isoform-specific attachment of osteoprogenitors to laminins: mapping to the short arms of laminin-1.

The recruitment of osteoblast progenitors involves their migration and attachment to the sites of bone formation through interactions with matrix proteins. In a time-limited cell attachment assay, coated laminin-1 inhibits the adhesion of most rat calvaria cells but attaches specifically to osteoprogenitors, as quantified by the number of bone colonies (nodules) formed in the cultures. In order to determine the molecular mechanisms involved in osteoprogenitor attachment to laminin-1, we investigated the effects of laminin-5, a N-truncated laminin variant. In contrast to laminin-1, laminin-5 increased (1.5-fold) rat calvaria cell attachment and did not display any specific affinity for osteoprogenitors. In competition experiments on laminin-5, blocking antibodies directed against either the integrin chain beta1 or the C-terminal portion of laminin-5, as well as thermic denaturation of the protein at 80 degrees C, inhibited rat calvaria cell attachment, suggesting the implication of integrin alpha3beta1 binding to the conformation-dependent C-terminal end of laminin-5. Stepwise thermic denaturation did not suppress the anti-adhesive activity of laminin-1, while osteoprogenitor recruitment was abolished after denaturation above 60 degrees C, suggesting that different domains are involved in these two effects. The anti-beta1 antibody further decreased RC cell attachment to laminin-1, providing evidence for concomitant anti-adhesive and beta1-dependent cell attachment activities. Blocking of beta1 integrin subunit did not, however, reduce osteoprogenitor recruitment. Finally, purified elastase digestion fragment E1+, encompassing the N-terminal short arms of laminin-1, reproduced the effects of the complete molecule in the assay, while C-terminal fragment E8 did not display any cell attachment or osteoprogenitor recruitment properties. In conclusion, the anti-adhesive and osteoprogenitor-selective effects of laminin-1 on rat calvaria cell populations are distinct, beta1-integrin-independent properties mapping to the short arms of the molecule and thus not displayed by the truncated laminin-5.

Selective attachment of osteoprogenitors to laminin.

During endochondral ossification and bone remodeling, osteoprogenitors (OP) attach to the matrix and differentiate into osteoblasts. To identify matrix proteins binding specifically these precursors, fetal rat calvaria (RC) cells were plated for 5-20 min in serum-free medium, on wells coated with various proteins and saturated with bovine serum albumin (BSA) to block nonspecific binding sites. Adherent cells were either counted or grown to assess bone colony (nodule) formation. As each nodule originates from the clonal division of one OP, the ratio (nodules/100 cells attached) measures the proportion of OP among adherent cells. Of numerous purified matrix proteins tested, laminin-1 and tenascin inhibited cell attachment, whereas fibronectin, bone sialoprotein, and type I collagen increased cell attachment and others had no effect. Only laminin-1 and, to a lesser extent, tenascin, enriched the cell population in OP. Laminin-1 acted time- and dose-dependently. In experiments in which cell attachment to laminin-coated but unsaturated wells was ensured by plating for 24 h in 10% fetal calf serum, laminin-1 had no effect on cell attachment nor on OP differentiation. In contrast, repeated plating of RC cells on laminin-1-coated/saturated wells depleted the population in OP, confirming that OP selection was a cell-attachment effect. When RC cell populations isolated by successive collagenase extractions were compared, the highest rate of OP enrichment on laminin-1 was obtained with the earliest populations, which were the most responsive to dexamethasone, a marker of early OP stages. In conclusion, laminin-1 recruits in vitro, through a cell-attachment effect, OP present in early RC cell populations, of which laminins are abundant extracellular matrix components.

LIF, but not IL-6, regulates osteoprogenitor differentiation in rat calvaria cell cultures: modulation by dexamethasone.

Cytokines of the interleukin 6 (IL-6) subfamily are a group of factors produced by osteoblasts and acting through the same transducing element, membrane protein gp130. We have previously shown that exogenous (added to the culture medium) leukemia inhibitory factor (LIF) inhibits bone nodule formation and expression of osteoblast-associated genes in fetal rat calvaria (RC) cell cultures and that dexamethasone (Dex) increases the ID50 of LIF. To investigate the respective roles of IL-6-related cytokines and receptors in osteprogenitor differentiation, and their regulatory interplay with Dex, we used reverse transcribed polymerase chain reaction, bioassay, and blocking antibody techniques to assess the time courses of LIF, IL-6, LIF transmembrane receptor, IL-6 receptor, and gp130 expression in RC cell cultures grown with and without Dex. The levels of the mRNAs for IL-6, LIF, and gp130 decreased concomitantly with the formation of bone nodules. Dex treatment, which stimulates bone nodule formation, reduced the expression of LIF and IL-6 mRNAs and IL-6 bioactivity in the culture medium. LIF treatment strongly stimulated the expression of IL-6. Incubation with anti-LIF antibodies increased the number of nodules, while an antibody blocking IL-6 activity had little or no effect on nodule numbers and did not antagonize the action of exogenous LIF, indicating that IL-6 does not mediate the action of LIF in this system. Moreover, although exogenously added IL-6 was active in the cultures as noted by a reduction of nodule mineralization, it had no effect on nodule numbers, i.e., on osteoprogenitor differentiation, in the presence or absence of Dex. In conclusion, IL-6, LIF, and their receptors are expressed throughout the time-course of osteogenesis in RC cell cultures. However, only LIF, but not IL-6, appears to play a significant role in autocrine regulation of osteoblastic differentiation in this system. The antagonist action of Dex on the effects of exogenously added LIF, as well as the bone-promoting action of Dex in RC cell cultures, could be exerted partly through the down-regulation of the expression of endogenous LIF.

Decorin inhibits cell attachment to thrombospondin-1 by binding to a KKTR-dependent cell adhesive site present within the N-terminal domain of thrombospondin-1.

Skin decorin (DCN) is an antiadhesive dermatan sulfate-rich proteoglycan that interacts with thrombospondin-1 (TSP) and inhibits fibroblast adhesion to TSP [Winnemöller et al., 1992]. Molecular mechanisms by which DCN interacts with TSP and inhibits cell adhesion to TSP are unknown. In the present study, we showed that skin DCN and bone DCN (chondroitin sulfate-rich proteoglycan) were quantitatively identical with respect to their ability to interact with TSP. Using a series of fusion proteins corresponding to the different structural domains of TSP, binding of [125I]DCN to TSP was found to be dependent of the N-terminal domain and, to a lesser extent, of the type 1 repeats and the C-terminal domain of TSP. In addition, heparan sulfate drastically inhibited [125I]DCN binding to solid-phase adsorbed TSP (80% inhibition), suggesting that DCN could bind to the N-terminal domain of TSP through interaction with heparin-binding sequences. To address this question, a series of synthetic peptides, overlapping heparin-binding sequences ARKGSGRR (residues 22-29), KKTR (residues 80-83) and RLRIAKGGVNDN (residues 178-189), were synthesized and tested for their ability to interact with DCN. [125I]DCN interacted only with peptides VDAVRTEKGFLLLASLRQMKKTRGT and KKTRGTLLALERKDHS containing the heparin-binding consensus sequence KKTR. These peptides contained glycosaminoglycan-dependent and -independent binding sites because [125I]DCN binding to VDAVRTEKGFLLLASLRQMKKTRGT and KKTRGTLLALERKDHS was partially reduced upon removal of the glycosaminoglycan chain (65% and 46% inhibition, respectively). [125I]DCN poorly bound to subpeptide MKKTRG and did not bind at all to subpeptides VDAVRTEKGFLLLASLRQ and TLLALERKDHS, suggesting that heparin-binding sequence MKKTRG constituted a DCN binding site when flanked with peptides VDAVRTEKGFLLLASLRQ and TLLALERKDHS. The sequence VDAVRTEKGFLLLASLRQMKKTRGTLLALERKDHS constitutes a cell adhesive active site in the N-terminal domain of TSP [Clezardin et al., 1997], and DCN inhibited the attachment of fibroblastic and osteoblastic cells to peptides VDAVRTEKGFLLLASLRQMKKTRGT and KKTRGTLLALERKDHS by about 50 and 80%, respectively. Although fibroblastic cells also attached to type 3 repeats and the C-terminal domain of TSP, DCN only inhibited cell attachment to the C-terminal domain. Overall, these data indicate that modulation by steric exclusion of cell adhesion to a KKTR-dependent cell adhesive site present within the N-terminal domain of TSP could explain the antiadhesive properties of DCN.

Osteonectin (SPARC) expression in human liver and in cultured human liver myofibroblasts.

Osteonectin/SPARC is a glycoprotein involved in the regulation of cell shape, adhesion, migration, and proliferation. It also has complex effects on extracellular matrix synthesis and turnover. We found that osteonectin mRNA was very abundant in a human liver myofibroblast library. Using Northern and Western blot, immunoprecipitation, and radioimmunoassay, we found that cultured liver myofibroblasts actively secreted osteonectin. Myofibroblasts are very rare in normal liver but proliferate during liver fibrosis where they synthesize extracellular matrix components. Thus, we studied the distribution of osteonectin in normal and fibrotic human liver using in situ hybridization. Osteonectin mRNA expression was weak in normal liver but very high in fibrotic liver within fibrous septae and scattered sinusoidal cells. Serial sectioning and double staining experiments with an antibody to smooth muscle alpha-actin showed that osteonectin transcripts were mostly co-localized with myofibroblasts. In conclusion, osteonectin is highly expressed in human liver myofibroblasts in culture as well as in human liver fibrosis in vivo. The many biological properties of osteonectin make it a candidate effector of human liver fibrogenesis.

The T3R alpha gene encoding a thyroid hormone receptor is essential for post-natal development and thyroid hormone production.

The diverse functions of thyroid hormones are thought to be mediated by two nuclear receptors, T3R alpha1 and T3R beta, encoded by the genes T3R alpha and T3R beta respectively. The T3R alpha gene also produces a non-ligand-binding protein T3R alpha2. The in vivo functions of these receptors are still unclear. We describe here the homozygous inactivation of the T3R alpha gene which abrogates the production of both T3R alpha1 and T3R alpha2 isoforms and that leads to death in mice within 5 weeks after birth. After 2 weeks of life, the homozygous mice become progressively hypothyroidic and exhibit a growth arrest. Small intestine and bones showed a strongly delayed maturation. In contrast to the negative regulatory function of the T3R beta gene on thyroid hormone production, our data show that the T3R alpha gene products are involved in up-regulation of thyroid hormone production at weaning time. Thus, thyroid hormone production might be balanced through a positive T3R alpha and a negative T3R beta pathway. The abnormal phenotypes observed on the homozygous mutant mice strongly suggest that the T3R alpha gene is essential for the transformation of a mother-dependent pup to an 'adult' mouse. These data define crucial in vivo functions for thyroid hormones through a T3R alpha pathway during post-natal development.