Modulation of PDGF mediated osteoblast chemotaxis by leukemia inhibitory factor LIF

Cell migration is a key event in tissue repair and remodeling. PDGF, a growth factor for multiple target cells, has been shown to be a potent chemoattractant for a variety of mesenchymal cells. However, it is likely that PDGF-mediated cell migration will be influenced by other cytokines that can be produced during physiological and pathological conditions. Leukemia inhibitory factor (LIF), a cytokine that is produced by a variety of cells including osteoblasts, may promote bone formation, but the mechanism is not known. Since osteoblasts are responsible for laying down new matrix during skeletal remodeling, in this report we have examined whether PDGF or LIF influences the migration of osteoblasts. Among several cytokines and growth factors tested, only PDGF was able to elicit a major chemotactic (directed migration) and a minor chemokinetic (random-migration) response in osteoblasts. LIF alone was not active in either chemotaxis or chemokinesis but when included with PDGF it caused a reduction in chemokinesis. Further, pretreatment of osteoblasts with LIF caused an increase in PDGF-driven chemotaxis. Finally, osteoblasts exposed briefly to LIF synthesized a higher level of non-collagenous proteins upon further treatment with PDGF. These observations are consistent with a role for LIF in promoting bone formation, both by influencing directional migration of osteoblasts and in laying down new matrix. © 1996 Wiley-Liss, Inc.     

The Wnt antagonist secreted frizzled-related protein-1 controls osteoblast and osteocyte apoptosis

Mechanisms controlling human bone formation remain to be fully elucidated. We have used differential display-polymerase chain reaction analysis to characterize osteogenic pathways in conditionally immortalized human osteoblasts (HOBs) representing distinct stages of differentiation. We identified 82 differentially expressed messages and found that the Wnt antagonist secreted frizzled-related protein (sFRP)-1 was the most highly regulated of these. Transient transfection of HOBs with sFRP-1 suppressed canonical Wnt signaling by 70% confirming its antagonistic function in these cells. Basal sFRP-1 mRNA levels increased 24-fold during HOB differentiation from pre-osteoblasts to pre-osteocytes, and then declined in mature osteocytes. This expression pattern correlated with levels of cellular viability such that the pre-osteocytes, which had the highest levels of sFRP-1 mRNA, also had the highest rate of cell death. Basal sFRP-1 mRNA levels also increased 29-fold when primary human mesenchymal stem cells were differentiated to osteoblasts supporting the developmental regulation of the gene. Expression of sFRP-1 mRNA was induced 38-fold following prostaglandin E2 (PGE2) treatment of pre-osteoblasts and mature osteoblasts that had low basal message levels. In contrast, sFRP-1 expression was down-regulated by as much as 80% following transforming growth factor (TGF)-beta1 treatment of pre-osteocytes that had high basal mRNA levels. Consistent with this, treatment of pre-osteoblasts and mature osteoblasts with PGE(2) increased apoptosis threefold, while treatment of pre-osteocytes with TGF-beta1 decreased cell death by 50%. Likewise, over-expression of sFRP-1 in HOBs accelerated the rate of cell death threefold. These results establish sFRP-1 as an important negative regulator of human osteoblast and osteocyte survival.     

BMP-2, BMP-4, and PDGF-bb stimulate chemotactic migration of primary human mesenchymal progenitor cells

For bone development, remodeling, and repair; the recruitment of mesenchymal progenitor cells (MPC) and their differentiation to osteoblasts is mandatory. The process of migration is believed to be regulated in part by growth factors stored within the bone matrix and released by bone resorption. In this study, primary human MPCs and to osteoblasts differentiated progenitor cells were examined for chemotaxis in response to human basic fibroblast growth factor (rhbFGF), human transforming growth factor beta 1 (rhTGF-beta1), human platelet derived growth factor bb (rhPDGF-bb), human bone morphogenetic protein-2 (rhBMP-2), and recombinant bone morphogenetic protein-4 of Xenopus laevis (rxBMP-4) from 0.001 to 1.0 ng/ml each. The results of migration were expressed as a chemotactic index (CI). Migration of primary human progenitor cells was stimulated by rhBMP-2, rxBMP-4, and rhPDGF-bb in a dose-dependent manner. The increase of CI was up to 3.5-fold for rhBMP-2, 3.6-fold for rxBMP-4, and up to 22-fold for rhPDGF-bb, whereas rhTGF-beta1 and rhbFGF did not stimulate cell migration in the concentration range tested. In contrast differentiated progenitor cells behave similar to primary human osteoblasts. RhBMP-2, rhPDGF-bb, and rhTGF-beta1 stimulated the migration from 2.2 to 2.4-fold each, while rxBMP-4 and rhbFGF reached only a CI of 1.7-1.6. The effect of rhBMP-2, rxBMP-4, and rhPDGF-bb as chemoattractive proteins for primary human MPC, including the change in response to growth factors after differentiation suggests a functional role for recruitment of MPCs during bone development and remodeling, as well as fracture healing.     

IL-1β Inhibits Human Osteoblast Migration

Bone has a high capacity for self-renewal and repair. Prolonged local secretion of interleukin 1β (IL-1β), however, is known to be associated with severe bone loss and delayed fracture healing. Since induction of bone resorption by IL-1β may not sufficiently explain these pathologic processes, we investigated, in vitro, if and how IL-1β affects migration of multipotent mesenchymal stromal cells (MSC) or osteoblasts. We found that homogenous exposure to IL-1β significantly diminished both nondirectional migration and site-directed migration toward the chemotactic factors platelet-derived growth factor (PDGF)-BB and insulinlike growth factor 1 (IGF-1) in osteoblasts. Exposure to a concentration gradient of IL-1β induced an even stronger inhibition of migration and completely abolished the migratory response of osteoblasts toward PDGF-BB, IGF-1, vascular endothelial growth factor A (VEGF-A) and the complement factor C5a. IL-1β induced extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinases (JNK) activation and inhibition of these signaling pathways suggested an involvement in the IL-1β effects on osteoblast migration. In contrast, basal migration of MSC and their migratory activity toward PDGF-BB was found to be unaffected by IL-1β. These results indicate that the presence of IL-1β leads to impaired recruitment of osteoblasts which might influence early stages of fracture healing and could have pathological relevance for bone remodeling in inflammatory bone disease.     

Immunohistochemical study of osteopontin expression during distraction osteogenesis in the rat.

Distraction osteogenesis (DO) is a limb-lengthening procedure that combines mechanical tension stress with fracture healing to provide a unique opportunity for detailed histological examination of bone formation. Osteopontin (OPN) is a multifunctional matricellular protein believed to play a key role in wound healing and cellular response to mechanical stress. We studied the expression of OPN during DO using standard immunohistochemical (IHC) staining techniques. In addition, we compared the expression of OPN to proliferation (PCNA-positive cells) in the DO gap. After 14 days of distraction in the rat, these stains revealed variations in OPN expression and its relationship to proliferation according to the cell type, tissue type, and mode of ossification examined. Fibroblast-like cells within the central fibrous area exhibited intermittent low levels of OPN, but no relationship was observed between OPN and proliferation. In areas of transchondral ossification, OPN expression was very high in the morphologically intermediate oval cells. During intramembranous ossification, osteoblasts appeared to exhibit a bimodal expression of OPN. Specifically, proliferating pre-osteoblasts expressed osteopontin, but OPN was not detected in the post-proliferative pre-osteoblasts/osteoblasts that border the new bone columns. Finally, intracellular OPN was detected in virtually all of the mature osteoblasts/osteocytes within the new bone columns, while detection of OPN in the matrix of the developing bone columns may increase with the maturity of the new bone. These results imply that the expression of OPN during DO may be more similar to that seen during embryogenesis than would be expected from other studies. Furthermore, the biphasic expression of OPN during intramembranous ossification may exemplify the protein's multi-functional role. Early expression may facilitate pre-osteoblastic proliferation and migration, while the latter downregulation may be necessary for hydroxyapatite crystal formation.     

To go or not to go: Migration of human mesenchymal progenitor cells stimulated by isoforms of PDGF

The recruitment of mesenchymal progenitor cells (MPCs) and their subsequent differentiation to osteoblasts is mandatory for bone development, remodeling, and repair. To study the possible involvement of platelet-derived growth factor (PDGF) isoforms, primary human MPCs and osteogenic differentiated progenitor cells (dOB) were examined for chemotaxic response to homodimeric human platelet-derived growth factor AA, -BB, and heterodimeric PDGF-AB. The role of PDGF receptors was addressed by preincubation with PDGF receptor alpha and beta chain specific antibodies. Migration of MPCs, dOB, and primary osteoblasts (OB) was stimulated by the addition of rhPDGF-AA, rhPDGF-BB, and rhPDGF-AB. The effect was highest in MPCs and for rhPDGF-BB, and declining with osteogenic differentiation. Preincubation with the receptor alpha specific antibody decreased the CI to borderline values while pretreatment with the receptor beta specific antibody led to a complete loss of chemotactic response to PDGF isoforms. In control experiments, basal migration values and rhBMP-2 as well as rxBMP-4 induced chemotaxis of MPC were not influenced by the addition of receptor alpha or beta antibodies. Interestingly, without preincubation the parallel exposure of MPC to rhTGF-beta1 instantaneously leads to a selective loss of migratory stimulation by rhPDGF-AA. The chemotactic effect of PDGF isoforms for primary human MPCs and the influence of osteogenic differentiation suggest a functional role for recruitment of MPCs during bone development and remodeling. Moreover, these observations may be useful for novel approaches towards guided tissue regeneration or tissue engineering of bone.     

Differential regulation of platelet-derived growth factor stimulated migration and proliferation in osteoblastic cells

Osteoblastic migration and proliferation in response to growth factors are essential for skeletal development, bone remodeling, and fracture repair, as well as pathologic processes, such as metastasis. We studied migration in response to platelet-derived growth factor (PDGF, 10 ng/ml) in a wounding model. PDGF stimulated a twofold increase in migration of osteoblastic MC3T3-E1 cells and murine calvarial osteoblasts over 24-48 h. PDGF also stimulated a tenfold increase in 3H-thymidine (3H-TdR) incorporation in MC3T3-E1 cells. Migration and DNA replication, as measured by BrdU incorporation, could be stimulated in the same cell. Blocking DNA replication with aphidicolin did not reduce the distance migrated. To examine the role of mitogen-activated protein (MAP) kinases in migration and proliferation, we used specific inhibitors of p38 MAP kinase, extracellular signal regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). For these signaling studies, proliferation was measured by carboxyfluorescein diacetate succinimidyl ester (CFSE) using flow cytometry. Inhibition of the p38 MAP kinase pathway by SB203580 and SB202190 blocked PDGF-stimulated migration but had no effect on proliferation. Inhibition of the ERK pathway by PD98059 and U0126 inhibited proliferation but did not inhibit migration. Inhibition of JNK activity by SP600125 inhibited both migration and proliferation. Hence, the stimulation of migration and proliferation by PDGF occurred by both overlapping and independent pathways. The JNK pathway was involved in both migration and proliferation, whereas the p38 pathway was predominantly involved in migration and the ERK pathway predominantly involved in proliferation.     

Proliferation versus migration in platelet-derived growth factor signaling: the key role of endocytosis

It is common knowledge that platelet-derived growth factor (PDGF) is a critical regulator of mesenchymal cell migration and proliferation. Nevertheless, these two cellular responses are mutually exclusive. To solve this apparent contradiction, we studied the behavior of NIH3T3 fibroblasts in response to increasing concentrations of PDGF. We found that there is strong cell proliferation induction only with PDGF concentrations >5 ng/ml, whereas the cell migration response arises starting from 1 ng/ml and is negligible at higher PDGF concentrations. According to these phenotypic evidences, our data indicate that cells display a differential activation of the main signaling pathways in response to PDGF as a function of the stimulation dose. At low PDGF concentrations, there is maximal activation of signaling pathways linked to cytoskeleton rearrangement needed for cell motility, whereas high PDGF concentrations activate pathways linked to mitogenesis induction. Our results suggest a mechanism by which cells switch from a migrating to a proliferating phenotype sensing the increasing gradient of PDGF. In addition, we propose that the cell decision to proliferate or migrate relies on different endocytotic routes of the PDGF receptor in response to different PDGF concentrations.     

Sphingosine 1-phosphate as a regulator of osteoclast differentiation and osteoclast-osteoblast coupling

Sphingosine 1-phosphate (S1P), produced by sphingosine kinase (SPHK), acts both by intracellular and extracellular modes. We evaluated the role of SPHK1 and S1P in osteoclastogenesis using bone marrow-derived macrophage (BMM) single and BMM/osteoblast coculture systems. In BMM single cultures, the osteoclastogenic factor receptor activator of NF-kappaB ligand (RANKL) upregulated SPHK1 and increased S1P production and secretion. SPHK1 siRNA enhanced and SPHK1 overexpression attenuated osteoclastogenesis via modulation of p38 and ERK activities, and NFATc1 and c-Fos levels. Extracellular S1P had no effect in these cultures. These data suggest that intracellular S1P produced in response to RANKL forms a negative feedback loop in BMM single cultures. In contrast, S1P addition to BMM/osteoblast cocultures greatly increased osteoclastogenesis by increasing RANKL in osteoblasts via cyclooxygenase-2 and PGE(2) regulation. S1P also stimulated osteoblast migration and survival. The RANKL elevation and chemotactic effects were also observed with T cells. These results indicate that secreted S1P attracts and acts on osteoblasts and T cells to augment osteoclastogenesis. Taken together, S1P plays an important role in osteoclastogenesis regulation and in communication between osteoclasts and osteoblasts or T cells.     

Osteogenesis in vitro: from pre-osteoblasts to osteocytes

Murine calvariae pre-osteoblasts (MC3T3-E1), grown in a novel bioreactor, proliferate into a mineralizing 3D osteoblastic tissue that undergoes progressive phenotypic maturation into osteocyte-like cells. Initially, the cells are closely packed (high cell/matrix ratio), but transform into a more mature phenotype (low cell/matrix ratio) after about 5 mo, a process that recapitulates stages of bone development observed in vivo. The cell morphology concomitantly evolves from spindle-shaped pre-osteoblasts through cobblestone-shaped osteoblasts to stellate-shaped osteocyte-like cells interconnected by many intercellular processes. Gene-expression profiles parallel cell morphological changes, up-to-and-including increased expression of osteocyte-associated genes such as E11, DMP1, and sclerostin. X-ray scattering and infrared spectroscopy of contiguous, square centimeter-scale macroscopic mineral deposits are consistent with bone hydroxyapatite, showing that bioreactor conditions can lead to ossification reminiscent of bone formation. Thus, extended-term osteoblast culture (≤10 mo) in a bioreactor based on the concept of simultaneous growth and dialysis captures the full continuum of bone development otherwise inaccessible with conventional cell culture, resulting in an in vitro model of osteogenesis and a source of terminally differentiated osteocytes that does not require demineralization of fully formed bone.     

Migration of CD4 T cells and dendritic cells toward sphingosine 1-phosphate (S1P) is mediated by different receptor subtypes: S1P regulates the functions of murine mature dendritic cells via S1P receptor type 3

Dendritic cells (DCs) and lymphocytes are known to show a migratory response to the phospholipid mediator, sphingosine 1-phosphate (S1P). However, it is unclear whether the same S1P receptor subtype mediates the migration of lymphocytes and DCs toward S1P. In this study, we investigated the involvement of S1P receptor subtypes in S1P-induced migration of CD4 T cells and bone marrow-derived DCs in mice. A potent S1P receptor agonist, the (S)-enantiomer of FTY720-phosphate [(S)-FTY720-P], at 0.1 nM or higher and a selective S1P receptor type 1 (S1P(1)) agonist, SEW2871, at 0.1 muM or higher induced a dose-dependent down-regulation of S1P(1). The pretreatment with these compounds resulted in a significant inhibition of mouse CD4 T cell migration toward S1P. Thus, it is revealed that CD4 T cell migration toward S1P is highly dependent on S1P(1). Mature DCs, when compared with CD4 T cells or immature DCs, expressed a relatively higher level of S1P(3) mRNA. S1P at 10-1000 nM induced a marked migration and significantly enhanced the endocytosis of FITC-dextran in mature but not immature DCs. Pretreatment with (S)-FTY720-P at 0.1 microM or higher resulted in a significant inhibition of S1P-induced migration and endocytosis in mature DCs, whereas SEW2871 up to 100 microM did not show any clear effect. Moreover, we found that S1P-induced migration and endocytosis were at an extremely low level in mature DCs prepared from S1P(3)-knockout mice. These results indicate that S1P regulates migration and endocytosis of murine mature DCs via S1P(3) but not S1P(1).     

Effects of platelet-derived growth factor on human and mouse osteoblastic cells isolated from the trabecular bone surface.

We show here that purified platelet derived growth factor (PDGF) stimulates DNA synthesis in normal endosteal mouse and human osteoblastic cells isolated by selective migration from the trabecular bone surface. Maximum DNA synthesis as measured by (3H)-thymidine incorporation into DNA was increased at 50 ng/ml PDGF (48-72 hours). In both species, the effect of PDGF (25 ng/ml) was lower than the mitogenic effect of 10% FCS. We found that the mitogenic effect of PDGF on human trabecular cells decreased with the number of cell passages. DNA synthesis was increased about 4-fold by PDGF (25 ng/ml) in early passaged cells that expressed low basal growth rate and high osteocalcin production in basal conditions and in response to 1,25(OH)2 vitamin D, whereas DNA synthesis was increased 1.2 fold by PDGF in late passaged cells that showed high basal growth rate and low osteocalcin release in absence or presence of 1,25(OH)2D. PDGF alone had no effect on osteocalcin production. These results indicate that PDGF has mitogenic effect on normal mouse and human osteoblastic cells lining the trabecular bone surface and that the responsiveness to PDGF of human trabecular cells varies with the stage of differentiation.     

Pulse application of platelet-derived growth factor enhances formation of a mineralizing matrix while continuous application is inhibitory

Platelet-derived growth factor (PDGF) stimulates chemotaxis and proliferation of osteoblasts, and induces bone formation in vivo. To determine how PDGF might regulate these cells, the effect of PDGF on long-term mineralizing cultures of fetal rat osteoblastic cells was examined. Although PDGF increased cell proliferation in these cultures, continuous treatment with PDGF caused a dose-dependent decrease in mineralized nodule formation. When cells were treated with multiple, brief (1 day) exposures to PDGF at the osteoblast differentiation stage, there was a significant 50% increase in mineralized nodule area. Based on modulation of alkaline phosphatase activity it appears that longer-term exposure to PDGF reduces mineralized nodule formation largely by inhibiting differentiated osteoblast function, while short-term exposure enhances proliferation without inhibiting the differentiated phenotype. Thus, the ultimate affect of PDGF on bone formation is likely to reflect two processes: a positive effect through enhancing cell number or a negative effect by inhibiting differentiated function. The inhibitory effect of PDGF on formation of a mineralized matrix is unlikely to be simply a result of enhanced proliferation of “fibroblastic” cells since cultures treated with PDGF for 3 days and then transferred to new plastic dishes exhibited a 70% increase in mineralized nodule area compared to controls. These results would predict that multiple, brief exposures to PDGF would enhance bone formation in vivo, while prolonged exposure to PDGF, which is likely to occur in chronic inflammation, would inhibit differentiated osteoblast function and limit bone regeneration. J. Cell. Biochem. 69:169–180, 1998. © 1998 Wiley-Liss, Inc.     

Sphingosine‐1‐phosphate (S1P) receptors: Promising drug targets for treating bone‐related diseases

Sphingosine‐1‐phosphate (S1P) is a natural bioactive lipid molecule and a common first or second messenger in the cardiovascular and immune systems. By binding with its receptors, S1P can serve as mediator of signalling during cell migration, differentiation, proliferation and apoptosis. Although the predominant role of S1P in bone regeneration has been noted in many studies, this role is not as well‐known as its roles in the cardiovascular and immune systems. In this review, we summarize previous research on the role of S1P receptors (S1PRs) in osteoblasts and osteoclasts. In addition, S1P is regarded as a bridge between bone resorption and formation, which brings hope to patients with bone‐related diseases. Finally, we discuss S1P and its receptors as therapeutic targets for treating osteoporosis, inflammatory osteolysis and bone metastasis based on the biological effects of S1P in osteoclastic/osteoblastic cells, immune cells and tumour cells.     

Cellular and molecular alterations of osteoblasts in human disorders of bone formation

Osteogenesis is a complex process characterized sequentially by the commitment of precursor cells, the proliferation of osteoprogenitor cells, the differentiation of pre-osteoblasts into mature osteoblasts and the apposition of a calcified bone matrix. Recent advances in cell and molecular biology have improved our knowledge of the cellular and molecular mechanisms controlling the different steps of bone formation in humans. Using ex vivo/in vitro studies of disorders of bone formation, we showed that the recruitment of osteoprogenitor cells is the most important step controlling the rate of bone formation in both rodents and humans. Accordingly, treatments stimulating osteoblast recruitment were found to increase bone formation in experimental models of osteopenic disorders. Using models of human osteoblastic cells, we identified the profile of phenotypic markers expressed during osteoblast differentiation, and found that hormones and growth factors control osteoblastic cell proliferation and differentiation in a sequential and coordinate manner during osteogenesis in vitro. Our recent evaluation of the phenotypic osteoblast abnormalities induced by genetic mutations in the Gs alpha and FGFR-2 genes led to the characterization of the role of these genes in the alterations of osteoblast proliferation and differentiation in humans. These studies at the histological, cellular and molecular levels provided new insight into the mechanisms that are involved in pathological bone formation in humans. It is expected that further determination of the pathogenic pathways in metabolic and genetic abnormalities in human osteoblasts will help to identify novel target genes and to conceive new therapeutic tools to stimulate bone formation in osteopenic disorders.     

Sphingosine 1-Phosphate (S1P) Receptors 1 and 2 Coordinately Induce Mesenchymal Cell Migration through S1P Activation of Complementary Kinase Pathways

Normal bone turnover requires tight coupling of bone resorption and bone formation to preserve bone quantity and structure. With aging and during several pathological conditions, this coupling breaks down, leading to either net bone loss or excess bone formation. To preserve or restore normal bone metabolism, it is crucial to determine the mechanisms by which osteoclasts and osteoblast precursors interact and contribute to coupling. We showed that osteoclasts produce the chemokine sphingosine 1-phosphate (S1P), which stimulates osteoblast migration. Thus, osteoclast-derived S1P may recruit osteoblasts to sites of bone resorption as an initial step in replacing lost bone. In this study we investigated the mechanisms by which S1P stimulates mesenchymal (skeletal) cell chemotaxis. S1P treatment of mesenchymal (skeletal) cells activated RhoA GTPase, but this small G protein did not contribute to migration. Rather, two S1P receptors, S1PR1 and S1PR2, coordinately promoted migration through activation of the JAK/STAT3 and FAK/PI3K/AKT signaling pathways, respectively. These data demonstrate that the chemokine S1P couples bone formation to bone resorption through activation of kinase signaling pathways.     

Sphingosine 1-phosphate and platelet-derived growth factor (PDGF) act via PDGF beta receptor-sphingosine 1-phosphate receptor complexes in airway smooth muscle cells

Platelet-derived growth factor (PDGF) and sphingosine 1-phosphate (S1P) act via PDGF beta receptor-S1P(1) receptor complexes in airway smooth muscle cells to promote mitogenic signaling. Several lines of evidence support this conclusion. First, both receptors were co-immunoprecipitated from cell lysates with specific anti-S1P(1) antibodies, indicating that they form a complex. Second, treatment of airway smooth muscle cells with PDGF stimulated the phosphorylation of p42/p44 MAPK, and this phosphorylated p42/p44 MAPK associates with the PDGF beta receptor-S1P(1) receptor complex. Third, treatment of cells with antisense S1P(1) receptor plasmid construct reduced the PDGF- and S1P-dependent activation of p42/p44 MAPK. Fourth, S1P and/or PDGF induced the formation of endocytic vesicles containing both PDGF beta receptors and S1P(1) receptors, which was required for activation of the p42/p44 MAPK pathway. PDGF does not induce the release of S1P, suggesting the absence of a sequential mechanism. However, sphingosine kinase 1 is constitutively exported from cells and supports activation of p42/p44 MAPK by exogenous sphingosine. Thus, the presentation of sphingosine from other cell types and its conversion to S1P by the kinase exported from airway smooth muscle cells might enable S1P to act with PDGF on the PDGF beta receptor-S1P(1) receptor complex to induce biological responses in vivo. These data provide further evidence for a novel mechanism for G-protein-coupled receptor and receptor tyrosine kinase signal integration that is distinct from the transactivation of receptor tyrosine kinases by G-protein-coupled receptor agonists and/or sequential release and action of S1P in response to PDGF.     

Sphingosine kinase-1/S1P1 signalling axis negatively regulates mitogenic response elicited by PDGF in mouse myoblasts

PDGF is known to be critically implicated in skeletal muscle repair; however its molecular mechanism of action has been only marginally investigated. In this study we show that in mouse myoblasts PDGF transactivates S1P₁ receptor via sphingosine kinase (SK)-1 activation and that this molecular event exerts a negative regulation of the mitogenic effect elicited by this growth factor. Indeed, pharmacological inhibition of S1P₁, or its specific silencing increased PDGF-dependent cell proliferation, whereas S1P₁ overexpression diminished the biological effect. Moreover, the mitogenic response to PDGF was enhanced by pharmacological inhibition of SK activity as well as specific silencing of SK1 but not SK2. Furthermore, ERK1/2 signalling pathway was found to be upstream of the observed attenuation of PDGF-induced cell proliferation. Interestingly, PDGF-directed engagement of S1P₁ exerted also a positive modulatory action of the growth factor-dependent cell motility. The here highlighted dual role of S1P₁-mediated signalling in response to myoblast challenge with PDGF is likely important to guarantee the fine control of the biological response to this growth factor, finalized to efficient repopulation of skeletal muscle after damage, where a tight balance between proliferation and migration of tissue progenitor cells is required.