Differential expression of FGF receptors and of myogenic regulatory factors in primary cultures of satellite cells originating from fast (EDL) and slow (Soleus) twitch rat muscles.

In the rat, the fast and slow twitch muscles respectively Extensor digitorum longus (EDL) and Soleus present differential characteristics during regeneration. This suggests that their satellite cells responsible for muscle growth and repair represent distinct cellular populations. We have previously shown that satellite cells dissociated from Soleus and grown in vitro proliferate more readily than those isolated from EDL muscle. Fibroblast growth factors (FGFs) are known as regulators of myoblast proliferation and several studies have revealed a relationship between the response of myoblasts to FGF and the expression of myogenic regulatory factors (MRF) of the MyoD family by myoblasts. Therefore, we presently examined the possibility that the satellite cells isolated from EDL and Soleus muscles differ in the expression of FGF receptors (FGF-R) and of MRF expression. FGF-R1 and -R4 were strongly expressed in proliferating cultures whereas FGF-R2 and R3 were not detected in these cultures. In differentiating cultures, only -R1 was present in EDL satellite cells while FGF-R4 was also still expressed in Soleus cells. Interestingly, the unconventional receptor for FGF called cystein rich FGF receptor (CFR), of yet unknown function, was mainly detected in EDL satellite cell cultures. Soleus and EDL satellite cell cultures also differed in the expression MRFs. These results are consistent with the notion that satellite cells from fast and slow twitch muscles belong to different types of myogenic cells and suggest that satellite cells might play distinct roles in the formation and diversification of fast and slow fibres.     

Basic fibroblast growth factor expression in human omental microvascular endothelial cells and the effect of phorbol ester.

The human omentum contains a potent, not yet identified angiogenic activity. The omentum is very vascularized. Therefore, we investigated whether human omental microvascular endothelial cells (HOME cells) express the angiogenic peptide basic fibroblast growth factor (bFGF). Cytosol prepared from HOME cells stimulated DNA synthesis in bovine epithelial lens cells (BEL cells). The mitogenic activity could be neutralized by an anti-bFGF antibody. Basic FGF-like material from the HOME cell cytosol was bound onto a heparin-Sepharose column at 0.6 M and was eluted at 3 M NaCl. The 3 M NaCl eluted material reacted with the specific anti-bFGF antibody in an ELISA and stimulated DNA synthesis. It did not react with a specific anti-acidic fibroblast growth factor (aFGF) antibody. Western blotting experiments using the same bFGF antibody showed the presence of a major band of 17 Kd and a doublet of 20-22 Kd. Northern blotting of non-stimulated HOME cells using a specific 1.4 kb bFGF probe showed the presence of 5 molecular species of 6.6, 3.7, 2.2, 2.0, and 1.0 kb. No aFGF mRNA was detected with a specific previously characterized 4.04 kb probe. 12-O-tetradecanoylphorbol 13-acetate (TPA) did not influence significantly the expression of bFGF at the protein and mRNA level in HOME cells. Thus, protein kinase C activation by TPA did not appear to modulate significantly the expression of bFGF for that cell type. Contrastingly, human umbilical vein endothelial cells (HUVE cells), which expressed no bFGF and aFGF mRNA at a basal level, were induced to express bFGF but not aFGF mRNA when stimulated by TPA. These results suggest that the described angiogenic activity could be the bFGF-like mitogen contained in HOME cells and that these cells are different from endothelial cells derived from large vessels (HUVE cells) regarding the expression of bFGF.     

Gene expression patterns of the fibroblast growth factors and their receptors during myogenesis of rat satellite cells.

Satellite cells are the myogenic precursors in postnatal muscle and are situated beneath the myofiber basement membrane. We previously showed that fibroblast growth factor 2 (FGF2, basic FGF) stimulates a greater number of satellite cells to enter the cell cycle but does not modify the overall schedule of a short proliferative phase and a rapid transition to the differentiated state as the satellite cells undergo myogenesis in isolated myofibers. In this study we investigated whether other members of the FGF family can maintain the proliferative state of the satellite cells in rat myofiber cultures. We show that FGF1, FGF4, and FGF6 (as well as hepatocyte growth factor, HGF) enhance satellite cell proliferation to a similar degree as that seen with FGF2, whereas FGF5 and FGF7 are ineffective. None of the growth factors prolongs the proliferative phase or delays the transition of the satellite cells to the differentiating, myogenin(+) state. However, FGF6 retards the rapid exit of the cells from the myogenin(+) state that routinely occurs in myofiber cultures. To determine which of the above growth factors might be involved in regulating satellite cells in vivo, we examined their mRNA expression patterns in cultured rat myofibers using RT-PCR. The expression of all growth factors, excluding FGF4, was confirmed. Only FGF6 was expressed at a higher level in the isolated myofibers and not in the connective tissue cells surrounding the myofibers or in satellite cells dissociated away from the muscle. By Western blot analysis, we also demonstrated the presence of FGF6 protein in the skeletal musle tissue. Our studies therefore suggest that the myofibers serve as the main source for the muscle FGF6 in vivo. We also used RT-PCR to analyze the expression patterns of the four tyrosine kinase FGF receptors (FGFR1-FGFR4) and of the HGF receptor (c-met) in the myofiber cultures. Depending on the time in culture, expression of all receptors was detected, with FGFR2 and FGFR3 expressed only at a low level. Only FGFR4 was expressed at a higher level in the myofibers but not the connective tissue cell cultures. FGFR4 was also expressed at a higher level in satellite cells compared to the nonmyogenic cells when the two cell populations were released from the muscle tissue and fractionated by Percoll density centrifugation. The unique localization patterns of FGF6 and FGFR4 may reflect specific roles for these members of the FGF signaling complex during myogenesis in adult skeletal muscle.     

Stretch-induced myogenin, MyoD, and MRF4 expression and acute hypertrophy in quail slow-tonic muscle are not dependent upon satellite cell proliferation

 The objectives of these studies were to determine if (1) hypertrophy-stimulated myogenic regulatory factor (MRF) mRNA increases occur in the absense of proliferating satellite cells, and (2) acute hypertrophy occurs without satellite cell proliferation. Adult and aged quails were exposed to 0 or 2500 Rads gamma irradiation, and then wing muscles were stretch-overloaded for 3 or 7 days. MRF mRNA levels in stretch-overloaded and contralateral anterior latissimus dorsi (ALD) muscles were determined after 3 days; hypertrophy was determined after 7 days. The elimination of proliferating cells in irradiated muscles was verified histologically by bromodeoxyuridine incorporation. Relative levels of MRF4, MyoD, and myogenin mRNA were elevated 100%–400% in stretch-overloaded ALD muscles from irradiated adult quails indicating that satellite cell proliferation was not a prerequisite for MRF mRNA increases. Myogenin was the only MRF that exhibited mRNA increases that were lowered by irradiation. This suggests that satellite cells contribute only to myogenin mRNA increases in non-irradiated adult muscles following 3 days of stretch-overload. Stretch-overloaded ALD muscles from aged quails had a relative increase in myogenin mRNA of ∼150%. The myogenin increase was the same in non-irradiated and irradiated aged animals and also the same as that in stretch-overloaded muscles from irradiated adult quails. Together, these data indicate that attenuated increases in MRF expression in muscles from aged animals are attributable to lower satellite cell MRF expression. ALD muscle masses and protein contents in adult irradiated quails approximately doubled after 7 days of stretch-overload demonstrating hypertrophy despite the elimination of satellite cell proliferation. Received: 5 June 1998 / Accepted: 19 November 1998     

Activation of myosin V-based motility and F-actin-dependent network formation of endoplasmic reticulum during mitosis

Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression, and were sorted as a CD34+ and CD45- fraction. Cells in this fraction were approximately 94% positive for Sca-1, and mostly negative (<3% positive) for CD14, 31, 49, 144, c-kit, and FLK-1. The CD34+/45- cells formed colonies in clonal cell cultures and colony-forming units displayed the potential to differentiate into adipocytes, endothelial, and myogenic cells. The CD34+/45- cells fully differentiated into vascular endothelial cells and skeletal muscle fibers in vivo after transplantation. Immediately after sorting, CD34+/45- cells expressed only c-met mRNA, and did not express any other myogenic cell-related markers such as MyoD, myf-5, myf-6, myogenin, M-cadherin, Pax-3, and Pax-7. However, after 3 d of culture, these cells expressed mRNA for all myogenic markers. CD34+/45- cells were distinct from satellite cells, as they expressed Bcrp1/ABCG2 gene mRNA (Zhou et al., 2001). These findings suggest that myo-endothelial progenitors reside in the interstitial spaces of mammalian skeletal muscles, and that they can potentially contribute to postnatal skeletal muscle growth.     

Inhibition of myogenic differentiation by fibroblast growth factor or type beta transforming growth factor does not require persistent c-myc expression

Skeletal muscle differentiation is accompanied by accumulation of the mRNA encoding the muscle isoenzyme of creatine kinase (MCK) and can be suppressed by serum components, fibroblast growth factor (FGF), or type beta transforming growth factor (TGF beta). Using the nonfusing myogenic cell line, BC3H1, the potential involvement of c-myc in growth factor-dependent inhibition of myogenesis was examined. Withdrawal of undifferentiated myoblasts from the cell cycle in medium with 0.5% serum was associated with a precipitous decline in expression of c-myc mRNA followed by induction of MCK mRNA. In 0.5% serum containing TGF beta, c-myc mRNA declined to a level identical to that in differentiated cells; however, MCK mRNA was not expressed. Exposure of quiescent differentiated cells to FGF or TGF beta caused disappearance of muscle-specific gene products and was accompanied by only transient low level induction of c-myc mRNA. These data indicate that persistent c-myc expression is not required for growth factor-mediated inhibition of myogenic differentiation.     

Characterization of myogenesis from adult satellite cells cultured in vitro

We describe several characteristics of in vitro myogenesis from adult skeletal muscle satellite cells from the rat and several amphibian species. The timing of cell proliferation and fusion into myotubes was determined, and in urodeles, myogenesis from satellite cells was clearly demonstrated for the first time. Growth factors are known to stimulate satellite cell proliferation. Acidic FGF mRNA was present in rat satellite cells during proliferation but it was not detected in myotubes. Fibronectin was synthesized in satellite cells during proliferation and expelled into the extracellular medium when the myotubes differentiated. We suggest that fibronectin plays a part in the formation of myotubes, as this process was inhibited by anti-fibronectin IgG. Adult satellite cells might differ from fetal myoblasts since they were observed to exhibit the opposite response to a phorbol ester (TPA) to that of the myoblasts. We therefore examined the possibility that the different levels of protein kinase C activity and different phorbol ester binding characteristics in the two cell types account for these opposite responses. Our results suggest that the difference is not connected with the phorbol ester receptor but might be caused by events subsequent to protein kinase C activation. Localized extracellular proteolytic activity might have a role in cell mobilization and/or fusion when satellite cells are activated. We showed that the content of plasminogen activators, chiefly urokinase, was larger in tissues from slow twitch muscles which regenerate more rapidly than fast muscles. The urokinase level rose sharply in cultures when cells fused into myotubes, and was twice as high in slow muscle cells as in fast ones. We also found that, in vitro, slow muscle satellite cells displayed greater myogenicity, but that phorbol ester inhibited their mitosis and myogenicity. We conclude that satellite cells acquire characteristics which differentiate them from myoblasts and correspond to the fast and slow muscles from which they originate.     

Muscle satellite cell-specific genes identified by genetic profiling of MyoD-deficient myogenic cell

Satellite cells are committed myogenic progenitors that give rise to proliferating myoblasts during postnatal growth and repair of skeletal muscle. To identify genes expressed at different developmental stages in the satellite cell myogenic program, representational difference analysis of cDNAs was employed to identify more than 50 unique mRNAs expressed in wild-type myoblasts and MyoD-/- myogenic cells. Novel expression patterns for several genes, such as Pax7, Asb5, IgSF4, and Hoxc10, were identified that were expressed in both quiescent and activated satellite cells. Several previously uncharacterized genes that represent putative MyoD target genes were also identified, including Pw1, Dapk2, Sytl2, and NLRR1. Importantly, many genes such as IgSF4, Neuritin, and Klra18 that were expressed exclusively in MyoD-/- myoblasts were also expressed by satellite cells in undamaged muscle in vivo but were not expressed by primary myoblasts. These data are consistent with a biological role for activated satellite cells that induce Myf5 but not MyoD. Lastly, additional endothelial and hematopoietic markers were identified supporting a nonsomitic developmental origin of the satellite cell myogenic lineage.     

The kinase inhibitor iso-H7 stimulates rat satellite cell differentiation through a non-protein kinase C pathway by increasing myogenin expression level

We analysed the signaling pathways involved in myogenic differentiation of primary cultures of rat satellite cells using substances targeting the protein kinase C (PKC) and the cAMP protein kinase (PKA) pathways. We have previously shown that iso-H7, which putatively inhibits both PKC and PKA, strongly stimulates satellite cell differentiation, as well as the PKA inhibitor HA 1004. In the study reported here, the effects of iso-H7 on satellite cell differentation were compared to those observed in the presence of agents which reduce PKC activity. It was shown that treatments with the highly specific PKC inhibitor GF109203X or with 12-O-tetradecanoylphorbol 13-acetate (TPA) which induced a partial PKC downregulation, did not significantly alter myogenic differentiation. Northern blot analyses showed that iso-H7 activated the expression of myogenin but not that of MyoD mRNA. Concurrently, iso-H7 increased myosin light-chain mRNA expression. In contrast, TPA had no effect on these syntheses. Taken together, these results showed that iso-H7 did not act intracellularly as a PKC inhibitor but rather as a PKA inhibitor as previously suggested. Our results are compatible with the hypothesis that a reduction in PKA activity controls satellite cell myogenesis through an increased myogenin mRNA expression.Abbreviations PKC protein kinase C - PKA cAMP-dependent protein kinase - CK creatine kinase - iso-H7 1-(5-isoquinolinesulfonyl)-3-methylpiperazine - H7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine - HA 1004 N-(3-hydroxyethyl)-1-piperazine ethanesulfonate - TPA 12-O-tetradecanoyl phorbol 13-acetate - MLC myosin light chain - GAPDH glyceraldehyde 3-phosphate deshydrogenase     

Differentially expressed fibroblast growth factors regulate skeletal muscle development through autocrine and paracrine mechanisms

Several FGF family members are expressed in skeletal muscle; however, the roles of these factors in skeletal muscle development are unclear. We examined the RNA expression, protein levels, and biological activities of the FGF family in the MM14 mouse skeletal muscle cell line. Proliferating skeletal muscle cells express FGF-1, FGF-2, FGF-6, and FGF-7 mRNA. Differentiated myofibers express FGF-5, FGF-7, and reduced levels of FGF-6 mRNA. FGF-3, FGF-4, and FGF-8 were not detectable by RT-PCR in either proliferating or differentiated skeletal muscle cells. FGF-I and FGF-2 proteins were present in proliferating skeletal muscle cells, but undetectable after terminal differentiation. We show that transfection of expression constructs encoding FGF-1 or FGF-2 mimics the effects of exogenously applied FGFs, inhibiting skeletal muscle cell differentiation and stimulating DNA synthesis. These effects require activation of an FGF tyrosine kinase receptor as they are blocked by transfection of a dominant negative mutant FGF receptor. Transient transfection of cells with FGF-1 or FGF-2 expression constructs exerted a global effect on myoblast DNA synthesis, as greater than 50% of the nontransfected cells responded by initiating DNA synthesis. The global effect of cultures transfected with FGF-2 expression vectors was blocked by an anti-FGF-2 monoclonal antibody, suggesting that FGF-2 was exported from the transfected cells. Despite the fact that both FGF-l and FGF-2 lack secretory signal sequences, when expressed intracellularly, they regulate skeletal muscle development. Thus, production of FGF-1 and FGF-2 by skeletal muscle cells may act as a paracrine and autocrine regulator of skeletal muscle development in vivo.     

Proliferating satellite cells express acidic fibroblast growth factor during in vitro myogenesis.

Recent in vitro studies have indicated that the proliferation of satellite cells, which are involved in muscular regeneration in vivo, is stimulated by exogenous addition of fibroblast growth factor (FGF). We present evidence that satellite cell cultures produce acidic, but not basic FGF. Acidic or basic FGF content was measured by enzyme immunoassay on cellular extracts after partial purification by heparin-Sepharose chromatography. During maximal cell proliferation, the level of acidic fibroblast growth factor (aFGF) was increased over fivefold from the values obtained before plating. aFGF content drastically dropped at the postmitotic stage to almost the threshold of detection, and remained weak as differentiation was completed. The immunolocalization of aFGF using highly purified anti-aFGF antibodies confirmed these results and indicated that aFGF was cytoplasma- or membrane-associated. Our work suggests that an endogenous production of aFGF by satellite cells may trigger cell proliferation by an intra- or autocrine mechanism, and therefore play an important role in muscular regeneration.     

Changes in proliferation,differentiation, fibroblast growth factor 2 responsiveness and expression of syndecan‐4 and glypican‐1 with turkey satellite cell age

Myogenic satellite cells are heterogeneous multipotential stem cells that are required for muscle repair, maintenance, and growth. The membrane‐associated heparan sulfate proteoglycans syndecan‐4 and glypican‐1 differentially regulate satellite cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) signal transduction, and expression of the myogenic regulatory factors MyoD and myogenin. The objective of the current study was to determine the effect of age on syndecan‐4 and glypican‐1 satellite cell populations, proliferation, differentiation, FGF2 responsiveness, and expression of syndecan‐4, glypican‐1, MyoD, and myogenin using satellite cells isolated from the pectoralis major muscle of 1‐day‐old, 7‐week‐old and 16‐week‐old turkeys. Proliferation was significantly reduced in the 16‐week‐old satellite cells, while differentiation was decreased in the 7‐week‐old and the 16‐week‐old cells beginning at 48 h of differentiation. Fibroblast growth factor 2 responsiveness was highest in the 1‐day‐old and 7‐week‐old cells during proliferation; during differentiation there was an age‐dependent response to FGF2. Syndecan‐4 and glypican‐1 satellite cell populations decreased with age, but syndecan‐4 and glypican‐1 were differentially expressed with age during proliferation and differentiation. MyoD and myogenin mRNA expression was significantly decreased in 16‐week‐old cells compared to the 1‐day‐old and 7‐week‐old cells. MyoD and myogenin protein expression was higher during proliferation in the 16‐week‐old cells and decreased with differentiation. These data demonstrate an age‐dependent effect on syndecan‐4 and glypican‐1 satellite cell subpopulations, which may be associated with age‐related changes in proliferation, differentiation, FGF2 responsiveness, and the expression of the myogenic regulatory factors MyoD and myogenin.     

Alternative splicing in fibroblast growth factor receptor 2 is associated with induced epithelial-mesenchymal transition in rat bladder carcinoma cells.

We described previously that acidic fibroblast growth factor (aFGF), but not basic fibroblast growth factor (bFGF), can induce the rat carcinoma cell line NBT-II to undergo a rapid and reversible transition from epithelial to mesenchymal phenotype (EMT). We now find that NBT-II EMT is stimulated by keratinocyte growth factor (KGF) in cells grown at low density. Accordingly, a high-affinity receptor showing 98% homology to mouse FGF receptor 2b/KGF receptor was cloned and sequenced from NBT-II cells. Northern analysis indicated that mRNA for FGF receptor 2b/KGF receptor was drastically down-regulated within 1 wk in aFGF-induced mesenchymal NBT-II cells. This decrease coincided with an up-regulation of FGF receptor 2c/Bek, a KGF-insensitive, alternatively spliced form of FGF receptor 2b/KGF receptor. Functional studies confirmed that KGF could not maintain EMT induction on mesenchymal NBT-II cells. FGF receptor 1 and FGF receptor 2c/Bek could also support EMT induction when transfected into NBT-II cells in response to aFGF or bFGF. Such transfected cells could bind bFGF as well as aFGF. Therefore, EMT can be induced through different FGF receptors, but EMT may also regulate FGF receptor expression itself.     

Human dermal fibroblasts express multiple bFGF and aFGF proteins

Summary We investigated the regulation of expression of bFGF and aFGF in cultures of normal human dermal fibroblasts grown in a defined, serum-free medium which did not contain FGF. Under these conditions we detected three molecular weight forms of bFGF protein [18.0, 23.0, and 26.6 kiloDaltons (kD)] and three molecular weight forms of aFGF protein (18.4, 19.2, and 28.6 kD) in these cells using western blot analysis. The addition of fetal bovine serum (FBS) to these cultures caused an accumulation of all three molecular weight forms of bFGF protein with a more dramatic accumulation of the 23.0 and 26.6 kD forms. In contrast, the addition of FBS to the cultures had no effect on the level of aFGF proteins. Analysis of mRNA isolated from cells grown in serum-free medium revealed multiple species of both bFGF and aFGF RNA with molecular weights that correlated with our previous observations. The abundance of all bFGF mRNA species increased dramatically after serum treatment while the abundance of aFGF mRNA species increased only slightly. Our observations demonstrate that factor(s) present in FBS elevate the levels of bFGF mRNA and protein beyond the levels already present in the cultures growing in serum-free medium. Moreover, both bFGF and aFGF protein are present in these cells as multiple molecular weight species. Some of these forms are higher in apparent molecular weight than would be predicted from ATG-initiated primary translation products of these genes. We also show that the cells used for this study proliferate in response to bFGF and aFGF, thus, it is possible that the growth of these cells could be subject to autocrine/paracrine control in certain conditions.