Focal contact clustering in osteoblastic cells under mechanical stresses: microgravity and cyclic deformation

We quantitatively compared vinculin-related adhesion parameters in osteoblastic cells submitted to two opposing mechanical stresses: low deformation and frequency strain regimens (stretch conditions) and microgravity exposure (relaxed conditions). In both ROS 17/2.8 cells and rat primary osteoblastic cells, 1% cyclic deformations at 0.05 Hz for 10 min per day for seven days stimulated cell growth compared to static culture conditions, while relaxed ROS cells proliferated in a similar way to static cultures (BC). We studied the short-term (up to 24 h) adaptation of focal contact reorganization under these two conditions. Cyclic deformation induced a biphasic response comprising the formation of new focal contacts followed by clustering of these focal contacts in both ROS cells and primary osteoblasts. Microgravity exposure induced a reduction in focal contact number and clustering in ROS cells. To evaluate whether the proliferation (stretch) or survival (relaxed) status of ROS cells influences focal contact organization, we inhibited the ERK proliferative-dependent pathway. Inhibition of proliferation by PD98059 was partially reversed, but not fully restored by stretch. Stretch-induced clustering of vinculin-positive contacts also persisted in the presence of PD98059, whereas the increase in focal contact number was abolished. In conclusion, we show that focal contacts are mechanoeffectors, and we suggest that their morphologic organization might serve as a discriminant functional parameter between survival and proliferation status in ROS 17/2.8 osteoblastic cells.     

Focal Contact Clustering in Osteoblastic Cells under Mechanical Stresses: Microgravity and Cyclic Deformation

We quantitatively compared vinculin-related adhesion parameters in osteoblastic cells submitted to two opposing mechanical stresses: low deformation and frequency strain regimens (stretch conditions) and microgravity exposure (relaxed conditions). In both ROS 17/2.8 cells and rat primary osteoblastic cells, 1% cyclic deformations at 0.05 Hz for 10 min per day for seven days stimulated cell growth compared to static culture conditions, while relaxed ROS cells proliferated in a similar way to static cultures (BC). We studied the short-term (up to 24 h) adaptation of focal contact reorganization under these two conditions. Cyclic deformation induced a biphasic response comprising the formation of new focal contacts followed by clustering of these focal contacts in both ROS cells and primary osteoblasts. Microgravity exposure induced a reduction in focal contact number and clustering in ROS cells. To evaluate whether the proliferation (stretch) or survival (relaxed) status of ROS cells influences focal contact organization, we inhibited the ERK proliferative-dependent pathway. Inhibition of proliferation by PD98059 was partially reversed, but not fully restored by stretch. Stretch-induced clustering of vinculin-positive contacts also persisted in the presence of PD98059, whereas the increase in focal contact number was abolished. In conclusion, we show that focal contacts are mechanoeffectors, and we suggest that their morphologic organization might serve as a discriminant functional parameter between survival and proliferation status in ROS 17/2.8 osteoblastic cells.     

Synthetic peptide GRGDS induces dissociation of alpha-actinin and vinculin from the sites of focal contacts

The synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) mimics the cellular binding site of many adhesive proteins in the extracellular matrix and causes rounding and detachment of spread cells. We have studied whether its binding affects the associations of two major components, alpha-actinin and vinculin, at the adhesion plaque. Living 3T3 cells were microinjected with fluorescently labeled alpha-actinin and/or vinculin and observed using video microscopy before and after the addition of 50 micrograms/ml GRGDS. As soon as 5 min after treatment, fluorescent alpha-actinin and vinculin became dissociated simultaneously from the sites of many focal contacts. The proteins either moved away as discrete structures or dispersed from adhesion plaques. As a result, the enrichment of alpha-actinin and vinculin at these focal contacts was no longer detected. The focal contacts then faded away slowly without showing detectable movement. These data suggest that the binding state of integrin has a transmembrane effect on the distribution of cytoskeletal components. The dissociation of alpha-actinin and vinculin from adhesion plaques may in turn weaken the contacts and result in rounding and detachment of cells.     

Calreticulin affects focal contact-dependent but not close contact-dependent cell-substratum adhesion.

We used two cell lines expressing fast (RPEfast) and slow (RPEslow) attachment kinetics to investigate mechanisms of cell-substratum adhesion. We show that the abundance of a cytoskeletal protein, vinculin, is dramatically decreased in RPEfast cells. This coincides with the diminished expression level of an endoplasmic reticulum chaperone, calreticulin. Both protein and mRNA levels for calreticulin and vinculin were decreased in RPEfast cells. After RPEfast cells were transfected with cDNA encoding calreticulin, both the expression of endoplasmic reticulum-resident calreticulin and cytoplasmic vinculin increased. The abundance of other adhesion-related proteins was not affected. RPEfast cells underexpressing calreticulin displayed a dramatic increase in the abundance of total cellular phosphotyrosine suggesting that the effects of calreticulin on cell adhesiveness may involve modulation of the activities of protein tyrosine kinases or phosphatases which may affect the stability of focal contacts. The calreticulin and vinculin underexpressing RPEfast cells lacked extensive focal contacts and adhered weakly but attached fast to the substratum. In contrast, the RPEslow cells that expressed calreticulin and vinculin abundantly developed numerous and prominent focal contacts slowly, but adhered strongly. Thus, while the calreticulin overexpressing RPEslow cells "grip" the substratum with focal contacts, calreticulin underexpressing RPEfast cells use close contacts to "stick" to it.     

Rotation in clinostat results in apoptosis of osteoblastic ROS 17/2.8 cells

Clinostat is an effective, ground-based tool which can be used to verify data from space flight, and to test hypotheses and experimental conditions for eventual space flights. Rotation in clinostat appears to mimic the microgravity environment by nulling the gravitational vector by continuous averaging. In the present study, we exposed osteoblast-like ROS 17/2.8 cells to a vector-averaged gravity environment in a clinostat and found that the cells undergo apoptotic death during the first 24 hr of clino-rotation. We suggest that apoptosis might be one of the mechanisms for reduced bone formation as observed in actual space flights.     

Protein tyrosine phosphatase-dependent proteolysis of focal adhesion complexes in endothelial cell apoptosis

Adenosine and/or homocysteine causes endothelial cell apoptosis, a mechanism requiring protein tyrosine phosphatase (PTPase) activity. We investigated the role of focal adhesion contact disruption in adenosine-homocysteine endothelial cell apoptosis. Analysis of focal adhesion kinase (FAK), paxillin, and vinculin demonstrated disruption of focal adhesion complexes after 4 h of treatment with adenosine-homocysteine followed by caspase-induced proteolysis of FAK, paxillin, and p130(CAS). No significant changes were noted in tyrosine phosphorylation of FAK or paxillin. Pretreatment with the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone prevented adenosine-homocysteine-induced DNA fragmentation and FAK, paxillin, and p130(CAS) proteolysis. Asp-Glu-Val-Asp-ase activity was detectable in endothelial cells after 4 h of treatment with adenosine-homocysteine. The PTPase inhibitor sodium orthovanadate did not prevent endothelial cell retraction or FAK, paxillin, or vinculin redistribution. Sodium orthovanadate did block adenosine-homocysteine-induced FAK, paxillin, and p130(CAS) proteolysis and Asp-Glu-Val-Asp-ase activity. Thus disruption of focal adhesion contacts and caspase-induced degradation of focal adhesion contact proteins occurs in adenosine-homocysteine endothelial cell apoptosis. Focal adhesion contact disruption induced by adenosine-homocysteine is independent of PTPase or caspase activation. These studies demonstrate that disruption of focal adhesion contacts is an early, but not an irrevocable, event in endothelial cell apoptosis.     

An in vitro model to evaluate cell adhesion to metals used in implantation shows significant differences between palladium and gold or platinum

Adhesion of tissue cells to metallic implants is a major factor that is important for proper tissue integration. Adhesion of Swiss mouse 3T3 fibroblasts to gold, platinum and palladium surfaces was investigated. Immunofluorescence staining for the integrin subunits alphav and beta1 and the focal contact protein vinculin revealed that cells growing on gold and platinum expressed many focal contacts. In contrast, cells on palladium surfaces had reduced numbers of focal contacts shown by vinculin staining and failed to demonstrate expression of alphav and beta1 in focal contacts. Spread cell area was also significantly reduced on palladium than on other surfaces suggesting that cells on palladium were more weakly attached. This may be due to either a different molecular composition of focal contacts in cells grown on palladium surfaces or unusual microstructural properties of the palladium surface. This model is useful to evaluate adhesion of cells to different metal surfaces.     

Contact-dependent regulation of vinculin expression in cultured fibroblasts: a study with vinculin-specific cDNA probes.

Vinculin specific cDNA clones were isolated from chicken embryo fibroblast (CEF) cDNA library in lambda gt11. The clones, ranging in size from 2.8 to 5.0 kb, were initially selected by rabbit antibodies to vinculin. Their identity was further confirmed by their specific reactivities with a battery of different vinculin-specific monoclonal antibodies. Southern blot analysis of restriction enzyme digested chicken spleen DNA suggested that all the isolated cDNA clones correspond to the same gene(s). Northern blot hybridization revealed that the vinculin-specific cDNA clones react with a single 6.5 kb mRNA in total cellular RNA preparations of CEF, whole chicken embryos and chicken gizzard smooth muscle. Moreover, fractionation of CEF poly(A)+ RNA by sucrose gradient centrifugation followed by translation in cell free system indicated that the mRNA coding for vinculin has a size of about 6.0-7.0 kb. The identity of these clones was finally confirmed by selection hybridization assay. The isolated vinculin-specific cDNA probes were subsequently used in order to study the effect of substrate adhesiveness on the expression of vinculin. We show here that cells cultured on highly adhesive substrate, such as endothelial extracellular matrix (ECM), form large vinculin-rich focal contacts, while cells grown on poorly adhesive substrate poly(2-hydroxyethyl methacrylate) [poly(HEMA)] contain only small distorted vinculin spots. These morphological differences were accompanied by over 5-fold reduction in vinculin synthesis in cells growing on poly(HEMA), compared to those cultured on the ECM and over 7.5-fold decrease in the levels of vinculin-specific mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts

We have recently observed a transmembrane association between extracellular fibronectin (FN) fibers and elongated focal patches or fibers of vinculin (VN) in G1-arrested stationary Nil 8 hamster fibroblasts, with double-label immunofluorescence microscopy (Singer and Paradiso, 1981, Cell. 24:481-492). We hypothesized that these FN-VN complexes might correspond to focal contacts, the membrane sites that are probably mainly responsible for attaching cells to their substrata, because vinculin is often localized in focal contacts. However, because fibronectin-vinculin associations may not be restricted to the substrate adhesive surface of the cell, it became necessary to determine whether some or all of the various kinds of FN-VN complexes which we described are in proximity to the substrate. Using interference reflection optics and double-label immunofluorescence microscopy for fibronectin and vinculin, many elongated (up to 38 micrometer) FN-VN associations were found to be strikingly coincident with focal contacts in the perinuclear area of extremely flattened arrested Nil 8 fibroblasts in 0.3% fetal bovine serum (FBS). In addition, the long FN-VN adhesion complexes were precisely aligned with the major phase-dense stress fibers observed at the ventral surfaces of these stationary cells with phase contrast microscopy. Fibronectin was neither associated with vinculin-containing focal contacts of Nil 8 cells cultured in medium with 5% FBS nor with vinculin-negative focal contacts located at the extreme edges of stationary cells arrested in 0.3 FBS. Our time-course experiments suggest that early FN-VN lacking- focal contacts, which form at the cellular margins, develop into mature substrate adhesion complexes containing both fibronectin and vinculin, localized in the major stress fibers at the centers of sessile fibroblasts.     

MAPK and SRC-kinases control EGR-1 and NF-kappa B inductions by changes in mechanical environment in osteoblasts

Bone loss occurs in microgravity whereas an increase in bone mass is observed after skeletal loading. This tissue adaptation involves changes in osteoblastic proliferation and differentiation whose mechanisms remain largely unknown. In this context, we investigated the expression and the nuclear translocation of Egr-1 and NF-kappa B, in a simulated microgravity model (clinostat) and in a model of mechanical strain (Flexcell). We performed RT-PCR and immunocytochemistry analyses at baseline and up to 2 h after stimulation (a mitogenic regimen, 1% stretch, 0.05 Hz, 10 min, or clinorotation 50 rpm, 10 min) in osteoblastic ROS17/2.8 cells. Egr-1 induction as well as NF-kappa B nuclear translocation were activated by mechanical changes. PKC downregulation and COX1/2 inhibition did not alter these inductions. In contrast, ERK1/2, p38(MAPK) and src-kinases pathways were differentially involved in both models. Thus, we demonstrated that changes in the mechanical environment induced an activation of Egr-1 and NF-kappa B with specific kinetics and involved various transduction pathways including MAPKs and src-kinases. These could partially explain the later alterations of proliferation observed.     

Inhibition of osteoblastic cell differentiation by conditioned medium derived from the human prostatic cancer cell line PC-3 in vitro

Human prostatic carcinoma frequently metastasizes to bone tissue and activates bone metabolism, especially bone formation, at the site of metastasis. It has been reported that an extract of prostatic carcinoma and conditioned medium (CM) of a human prostatic carcinoma cell line, PC-3, established from a bone metastastic lesion, stimulate osteoblastic cell proliferation. However, there is little information about the effect of PC-3 CM on the differentiation of osteoblastic cells. In this study, we investigated the effect of PC-3 CM on the differentiation of two types of osteoblastic cells, primary fetal rat calvaria (RC) cells containing many undifferentiated osteoprogenitor cells, and ROS 17/2.8, a well-differentiated rat osteosarcoma cell line. PC-3 CM inhibited bone nodule formation and the activity of alkaline phosphatase (ALPase), an osteoblastic marker enzyme, on days 7, 14, and 21 (RC cells) or 3, 6, and 9 (ROS 17/2.8 cells) in a dose-dependent manner (5–30% CM). However, the CM did not affect cell proliferation or cell viability. PC-3 CM was found to markedly block the gene expression of ALPase and osteocalcin (OCN) mRNAs but had no effect on the mRNA expression of osteopontin (OPN), the latter two being noncollagenous proteins related to bone matrix mineralization. These findings suggest that PC-3 CM contains a factor that inhibits osteoblastic cell differentiation and that this factor may be involved in the process of bone metastasis from prostatic carcinoma. J. Cell. Biochem. 67:248–256, 1997. © 1997 Wiley-Liss, Inc.     

Differential temperature sensitivity of cultured cells from cartilaginous or bone origin.

The effects of long-term exposure to hyperthermia were studied on several cell cultures of cartilaginous or bone origin after a 4-day treatment at 40 degrees C. Chondrocytes proliferation, as well as mitochondrial activity were not modified by these culture conditions (40 degrees C) but protein content and cell volume were increased. In contrast, the proliferative capacity of osteoblasts, MC3T3.E1 a and ROS 17/2.8 was decreased and their protein content, cell volume and mitochondrial activity were increased. Chondrocytes appeared to be thermoresistant, and osteoblastic cells thermosensitive. Furthermore, temperature sensitivity was greater for the continuous established osteoblastic cell line MC3T3.E1 and for the cancerous established osteoblastic cell line ROS 17/2.8 than for chondrocytes.     

Effects of GDF7/BMP12 on proliferation and alkaline phosphatase expression in rat osteoblastic osteosarcoma ROS 17/2.8 cells

Growth and differentiation factor 7(GDF7), also later called as bone morphogenetic protein (BMP)12, is a new member of the BMP superfamily, which induces formation of tendon-like tissue formation in the ectopic implantation experiments. We examined the effect of BMP12 on proliferation and expression of phenotype-related genes in rat osteoblastic osteosarcoma ROS17/2.8 cells. BMP12 treatment enhanced proliferation of ROS17/2.8 cells within 3 days and this effect was observed at least up to day 6 of the treatment. The cell number was increased by about 50% on day 3 and about two-fold by day 6. These effects were observed at the dose range between 40 and 1,000 ng/ml. Treatment with BMP12 also enhanced alkaline phosphatase activity by about 50% in ROS17/2.8 cells within 24 h of the treatment. The effect peaked at 48 h and was still observed at 72 h. The enhancing effect of BMP12 on alkaline phosphatase was observed similarly at the doses ranging from 40 to 1,000 ng/ml. These data indicate that BMP12 has positive effects on proliferation and phenotypic expression of ROS 17/2.8 cells.     

3-D surface charges modulate protrusive and contractile contacts of chondrosarcoma cells

Up to now, most of the studies addressing the critical roles played by protrusive and contractile cell-matrix contacts in cell adhesion, guidance, migration, matrix assembly, and activation of signaling molecules have been performed on two-dimensional surfaces. Here, we analysed the organization of chondrosarcoma cell contacts in a new three-dimensional environment made of titanium beads. Surface charges were modified by deposition of polyelectrolyte multilayer films built up by alternated polycations poly-(L-lysine) or poly(allylamine hydrochloride) and polyanions poly-(L-glutamic acid) or poly(sodium 4-styrenesulfonate). Negatively charged 3-D titanium surfaces amplified the occurrence and length of cell protrusions. These protrusions had pseudopod characteristics extended to 200 microm in length, growing off the substratum to distant beads. Pseudopod formation is inhibited by the exocytosis inhibitor concanamycin A and is triggered by a secreted factor. Chondrosarcoma cells adhering on uncoated or on negatively charged surfaces contained discrete focal spots of vinculin and actin cables. In cells plated onto these surfaces, phosphorylation of p44/42 MAPK/ERK was twofold increased. In contrast, no cytoskeletal vinculin and actin organization was observed when the surface was positively charged. These data suggest that chondrosarcoma cells adapt a more stable adhesion on uncoated or negatively charged surfaces. This point may be critical in tissue engineering strategies designed for cartilage repair.     

Organization of the cytoskeleton and the focal contacts of bovine aortic endothelial cells cultured on type I and III collagen.

We investigated the organization of the cytoskeleton and the focal contacts of bovine aortic endothelial cells cultured on type I and III collagen. The influence of these collagens on cell morphology and the distribution pattern of actin, vimentin, talin, and vinculin was analyzed by light microscopy, conventional electron microscopy, immunofluorescence, and immunogold labeling after lysis-squirting. Whereas the morphology of the endothelial cells is not markedly influenced, the structure of the cytoskeleton and the focal contacts of the cells are altered by the different collagen types. Stress fibers are more distinct in cells grown on type I collagen; cells on type III collagen show a more diffuse distribution of actin molecules. Intermediate filaments seem not to be affected by the collagens. The areas of focal contacts are larger in cells on type I collagen. Additionally, the labeling pattern of talin and vinculin is denser in focal contacts of cells grown on type I collagen. These results suggest an important role of the type of collagen in mediation of the organization of the microfilament system and the adhesion structures of bovine aortic endothelial cells in culture.     

MAP and src kinases control the induction of AP-1 members in response to changes in mechanical environment in osteoblastic cells

The activating protein-1 (AP-1) complex plays a critical role in bone physiology, including its response to strain. We studied gene expression and nuclear translocation kinetics of the seven AP-1 members, after substrate deformation (Flexcell) or simulated microgravity (Clinostat), in osteoblastic ROS17/2.8 cells. Gene expression and nuclear translocation of all the AP-1 members were induced, under both conditions, with differences in their kinetics, except fosB mRNA in the Clinostat. Downregulation of protein kinase C (PKC) and COX1/2 or inhibition of ERK1/2, p38(MAPK) or src kinases had no major effect on AP-1 mRNA expression in the Flexcell. In contrast, ERK1/2, p38(MAPK) and src kinases treatment blocked nuclear translocation of almost all the AP-1 members in both models, except Fra-1, JunD after deformation and Fra-1, JunB after clinorotation. Thus, changes in the osteoblastic mechanical environment induced a dramatic induction of most of the AP-1 members with specific kinetics and involved MAPK and src kinase pathways, which differed whether the cells were stretched or clinorotated.     

Role of vinculin in the maintenance of cell-cell contacts in kidney epithelial MDBK cells

Microinjection of fluorophore-tagged cytoskeletal proteins has been a useful tool in studies of formation of focal adhesions (FA). We used this method to study the maintenance of adherens junctions (AJ) and tight junctions (TJ) of epithelial Madin-Darby bovine kidney cells. We chose alpha-actinin and vinculin as markers, because they are present both at adherens junctions and focal adhesions and their binding partners have been well characterized. Isolated FITC-labelled chicken alpha-actinin and vinculin were injected into confluent cells where they were rapidly incorporated both in FAs and AJs. The FAs remained unchanged, whereas cell-cell contacts began to fade within an hour after injection and the cells were joined to polykaryons having 5 to 13 nuclei. Short fragments of cell membranes containing injected proteins, actin, beta-catenin, cadherin, claudin, occludin and ZO-1 were visible inside the polykaryons indicating that both AJs and TJs were disintegrated as a single complex. Microinjected FITC-labelled vinculin head domain was also incorporated to both AJs and FAs, but instead of fusions it rapidly induced the detachment of the cells from the substratum probably due to high affinity of vinculin head to talin. Vinculin tail domain had no apparent effect on the cell morphology. Since small GTPases are involved in the building up of AJs, we injected active and inactive forms of cdc42 and rac proteins together with vinculin to see their effect. Active forms reduced the formation of polykaryons presumably by strengthening AJs, whereas inactive forms had no apparent effect. We suggest that excess alpha-actinin and vinculin uncouple the cell-cell adhesion junctions from the intracellular cytoskeleton which leads to fragmentation of junctional complexes and subsequent cell fusion. The results show that cell-cell adhesion sites are more dynamic and more sensitive than FAs to an imbalance in the amount of free alpha-actinin and intact vinculin.     

Focal contacts: transmembrane links between the extracellular matrix and the cytoskeleton

The sites of tightest adhesion that form between cells and substrate surfaces in tissue culture are termed focal contacts. The external faces of focal contacts include specific receptors, belonging to the integrin family of proteins, for fibronectin and vitronectin, two common components of extracellular matrices. On the internal (cytoplasmic) side of focal contacts, several proteins, including talin and vinculin, mediate interactions with the actin filament bundles of the cytoskeleton. The changes that occur in focal contacts as a result of viral transformation are discussed.     

Adhesiveness and distribution of vinculin and spectrin in retinal pigmented epithelial cells during growth and differentiation in vitro

Colonies of chick retinal pigmented epithelial (RPE) cells offer an excellent model system for studying the organization of cytoskeleton in sheets of differentiating epithelial cells. The cells occupying the center of the colony resemble RPE cells in vivo and are cuboidal, pigmented, and relatively nonadherent while those toward the periphery gradually become flatter, nonpigmented, motile, and strongly adherent to the substratum. Immunofluorescence microscopy with antiserum against chicken erythrocyte alpha-spectrin reveals that this protein is present in the cortex of RPE cells in all parts of the colony. It is neither concentrated in, nor excluded from the regions occupied by the major microfilament bundles, and its distribution is not related to the adhesion patterns visualized by surface reflection interference microscopy. In contrast, the distribution of vinculin is closely correlated with the adhesiveness of RPE cells in different parts of the colony. Immunofluorescence microscopy reveals that in the RPE cells vinculin may be diffusely distributed in the cytoplasm; present in a cortical band outlining the cell borders; and present in focal contacts and adhesions. The distribution of vinculin is affected by the length of time the colonies grow in culture, by the degree of cell packing and by the adhesiveness of cells to the substratum. In RPE cells grown in vitro for short periods (less than or equal to 3 days) vinculin is found in focal contacts and adhesions in both the undifferentiated, well spread peripheral cells as well as in the differentiated, polygonally packed central cells of the colony. In RPE cells cultured for longer periods (greater than or equal to 14 days) vinculin is present in focal contacts and adhesions only in strongly adherent, undifferentiated cells at the edge of the colony. In packed central cells of both short- and long-term cultures vinculin is found in the cortical band which circumscribes the apical ends of cells at the level of the adherens type intercellular junctions. Its appearance in the cortical bands does not depend on the length of time the colonies are grown in vitro but on the presence of cell-cell contacts resulting from an increased degree of cell packing within the central part of the colony. These results are discussed in relation to the development and the role of extracellular matrix in determining the adhesiveness of RPE cells in vitro.     

Identification and activation of mitogen-activated protein (MAP) kinase in normal human osteoblastic and bone marrow stromal cells: Attenuation of MAP kinase activation by cAMP,parathyroid hormone and forskolin

The mitogen-activated protein (MAP) kinases (p44^mapk and p42^mapk), also known as extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), are activated in response to a variety of extracellular signals, including growth factors, hormones and, neurotransmitters. We have investigated MAP kinase signal transduction pathways in normal human osteoblastic cells. Normal human bone marrow stromal (HBMS), osteoblastic (HOB), and human (TE85, MG-63, SaOS-2), rat (ROS 17/2.8, UMR-106) and mouse (MC3T3-E1) osteoblastic cell lines contained immunodetectable p44^mapk/ERK1 and p42^mapk/ERK2. MAP kinase activity was measured by 'in-gel' assay myelin basic protein as the substrate. Mainly ERK2 was rapidly activated (within 10 min) by bFGF, IGF-I and PDGF-BB in normal HOB, HBMS and human osteosarcoma cells, whereas both ERK1 and ERK2 were activated by growth factors in rat osteoblast-like cell lines, ROS 17/2.8 and UMR-106. The ERK1 activation was greater than the ERK2 in ROS 17/2.8 cells. Furthermore, ERK2 was also activated by bFGF and PDGF-BB in the mouse osteoblastic cell line, MC3T3-E1. This is the first demonstration of inter-species differences in the activation of MAP kinases in osteoblastic cells. Cyclic AMP derivatives or cAMP generating agents such as PTH and forskolin inhibited ERK2 activation by bFGF and PDGF-BB suggesting a 'cross-talk' between the two different signalling pathways activated by receptor tyrosine kinases and cAMP-dependent protein kinase. The accumulated results also suggest that the MAP kinases may be involved in mediating mitogenic and other biological actions of bFGF, IGF-I and PDGF-BB in normal human osteoblastic and bone marrow stromal cells.