Denis Barritault

GROWTH FACTORS IN SKELETAL MUSCLE REGENERATION

Adult skeletal muscles are able to regenerate after injury. This process is due to the activation of quiescent muscle precursor cells, also called satellite cells, which proliferate and differentiate to form new myotubes. In this regeneration process, several growth factors which come from the muscle and/or from the motor nerve and inflammatory cells have been shown to play key roles. However, most of our knowledge comes from in vitro studies, where, during myogenesis, proliferation of satellite cells is regulated by FGFs, TGF beta s, PDGF, IGF-I and II, while differentiation appears to be promoted mainly by IGFs. During regeneration in vivo, most of these factors have been shown to operate and interact. Other factors also appear to condition the regeneration process, such as LIF, which acts predominantly as a proliferative factor; and HARP/PTN/HB-GAM and other neurotrophic factors, which may be necessary for the formation of new neuromuscular junctions. TGF beta has a major influence on the reorganisation of the extracellular matrix. This review presents a critical summary of the known effects of growth factors on skeletal muscle regeneration.

FGF-2 and TPA induce matrix metalloproteinase-9 secretion in MCF-7 cells through PKC activation of the Ras/ERK pathway.

Matrix metalloproteinases (MMPs) play an important role in cancer metastasis. Here, we investigated the effect of fibroblast growth factor-2 (FGF-2) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the secretion of type IV collagenases (MMP-2, MMP-9) in breast cancer MCF-7 cells. As shown by gelatin zymography, both FGF-2 and TPA stimulated the secretion of MMP-9 in MCF-7 cells while they did not change the level of MMP-2 secretion. Signaling cascade studies indicated that both FGF-2 and TPA induced Ras activation, c-Raf phosphorylation, mitogen-activated protein kinase/ERK kinase (MEK(1/2)) phosphorylation, and extracellular signal-regulated kinase (ERK(1/2)) phosphorylation. The FGF-2- and TPA-induced MMP-9 secretion was significantly inhibited by transient transfection of MCF-7 cells with dominant negative Ras (Ras-N17) and by treatment with MEK(1/2) inhibitor PD98059. A pan-protein kinase C (PKC) inhibitor, GF109203X, was found to totally abolish the FGF-2- and TPA-induced MMP-9 secretion and ERK(1/2) phosphorylation. Use of isoform-specific PKC inhibitors such as Rotllerin and Gö6976 suggested, moreover, that the PKC-delta isoform is a likely component of FGF-2 and TPA trophic signaling. These results demonstrated that FGF-2 and TPA induce MMP-9 secretion in MCF-7 cells mainly through PKC-dependent activation of the Ras/ERK(1/2) signaling pathway.

Specific fixation of bovine brain and retinal acidic and basic fibroblast growth factors to mouse embryonic eye basement membranes.

The labeling pattern of mouse embryonic eye frozen sections incubated with radioiodinated brain acidic and basic fibroblasts growth factors (aFGF and bFGF) was investigated by autoradiography. Both growth factors bind to basement membranes in a dose-dependent way, with a higher affinity for bFGF. Similar data were obtained with eye-derived growth factors (EDGF), the retinal forms of FGF. There was a heterogeneity in the affinity of the various basement membranes toward these growth factors. The inner limiting membrane of the retina and the posterior part of the lens capsule have a higher binding capacity than the posterior part of the Bruchs membrane. The specificity of the growth factor-basement membrane interaction was demonstrated by the following experiments: (i) an excess of unlabeled growth factor displaced the labeling; (ii) unrelated proteins with different isoelectric points--gelatin, serum albumin, histones--did not modify the labeling; and (iii) iodinated EGF or PDGF did not label basement membrane. In order to get a better understanding of the nature of this binding, we performed the incubation of the frozen sections with iodinated FGFs preincubated with various compounds: (i) heparin which is known to have a strong affinity for aFGF and bFGF partially decreases the labeling, and (ii) chondroitin sulfate B and dextran sulfate at high concentrations were also partially effective. In addition, enzymatic treatment of the sections reveals that only heparitinase, not collagenase or chondroitinase ABC, completely prevents the labeling without destroying the overall structure of the basement membrane. An antibody against the proteic part of EHS mouse proteoheparan sulfate does not affect the signal. Esterification of the acidic groups cancelled the binding. These results demonstrate that FGFs bind specifically to basement membranes, probably on the polysaccharidic part of the proteoheparan sulfate, and suggest that this type of interaction may be a general feature of the mechanism of action of these growth factors.

MCF7 mammary cancer cells respond to bFGF and internalize it following its release from extracellular matrix: a permissive role of cathepsin D.

High and low affinity receptors for basic fibroblast growth factor (bFGF) were detected by binding experiments on MCF7 breast cancer cells. These cells were stimulated for growth by physiological concentrations of bFGF. However, in contrast to endothelial cells, these MCF7 cells did not produce detectable amounts of biologically active bFGF or related heparin-binding growth factor(s) of the FGF family. In vitro, the cathepsin D (cath-D) secreted by MCF7 cells was able to digest extracellular matrix (ECM) and to release ECM-bound 125I-bFGF. When MCF7 cells were cultured on ECM containing bound bFGF, they internalized bFGF, which was slowly and partially proteolyzed in the cells. Processing occurred in acidic compartments and was inhibited by leupeptin. Pepstatin A, an inhibitor of aspartyl proteases, had no effect on the processing but reduced internalization of matrix-bound bFGF by MCF7 cells. Taken together, these results suggest a cooperation between cath-D and bFGF, by which the protease could facilitate the release of bFGF from ECM and its subsequent use by breast cancer cells and/or adjacent cells involved in angiogenesis.

Bovine retina contains three growth factor activities with different affinity to heparin: eye derived growth factor I, II, III

Several ocular tissues have been shown to contain growth factor activity designated under a generic name as Eye Derived Growth Factor. Purification from bovine retina was undertaken and a fraction which could induce target cells to proliferate at doses of 5 ng per ml of culture medium was obtained. Using heparin sepharose chromatography we now show that this mitogenic activity can be fractionated into three different activities. Crude extract of bovine retina used as starting material was separated into two major fractions, one with no affinity for heparin and which was named Eye Derived Growth Factor III, and one with a strong affinity for heparin and eluted from the column with 1.4 M NaCl named Eye Derived Growth Factor I. This fraction EDGF I induces cell proliferation at doses of 100 pg/ml of culture medium. A 10(5) fold purification was achieved by this single chromatography step. Cibacron Blue purified EDGF was also further fractionated by heparin sepharose. All biological activity was found to bind to heparin. One fraction eluted at 1 M NaCl named Eye Derived Growth Factor II had a biological activity at doses of 1 ng while the other growth factor was the EDGF I with biological activity at 25 pg. At this step of purification EDGF I runs as a single band on SDS polyacrylamide gel at a molecular weight of 17 000 d. These data strongly suggest that Eye Derived Growth Factors I and II are respectively similar to Brain Fibroblast Growth Factor and to Endothelial Cell Growth Factor from hypothalamus.

Mitogenic properties of a new endothelial cell growth factor related to pleiotrophin

A growth factor was isolated from a neutral pH extract of adult bovine brain. Purification of this polypeptide was achieved by a three step procedure including cationic exchange, heparin-Sepharose affinity and Mono S chromatography. This heparin binding protein had a molecular weight of 18,000 as assessed by silver-stained SDS-PAGE and was not immunologically and structurally related to acidic or basic FGF. Freshly purified protein had a maximal mitogenic effect on bovine brain capillary cells at a concentration of 100 pM. Microsequencing revealed an unique amino-terminal sequence homologous to heparin-binding growth-associated molecule (HB-GAM), a neuronal maturation protein, to pleiotrophin (PTN), a fibroblast cell growth factor and to one form of the putative protein product of the MK gene, a retinoic acid induced-gene.

Increased Immunodetection of Acidic Fibroblast Growth Factor in Bladder Cancer, Detectable in Urine

Acidic fibroblast growth factor is a regulatory peptide involved in cell proliferation, differentiation and motility. We used a polyclonal antiserum raised against purified native bovine acidic fibroblast growth factor, with no cross-reactivity for basic fibroblast growth factor to detect acidic fibroblast growth factor in tissue extracts and urine samples by means of a competitive enzyme immunoassay. Histochemical analysis was also performed on 10 specimens of normal urothelium and 50 of bladder cancer. Acidic fibroblast growth factor immunoreactive material was found in normal urothelium (1.77 +/- 2 ng./gm. tissue) and was increased more than 10-fold in patients with transitional cell carcinoma of the bladder (20.36 +/- 12 ng./gm. tissue). Immunohistochemical analysis localized immunoreactivity in the epithelial compartment of bladder tumors. Acidic fibroblast growth factor was assayed in urine from 579 individuals comprising a control group (114) and patients with benign prostatic hypertrophy (133), carcinoma of the prostate (96) or transitional cell carcinoma of the bladder (236). There was a significant difference in the frequency of urinary acidic fibroblast growth factor detection among the patients with invasive transitional cell carcinoma, the control group (p < 0.001) and the patients with prostatic disease (p < 0.01). The sensitivity was 72% and the specificity was 91%. Furthermore, the frequency of acidic fibroblast growth factor detection by enzyme immunoassay in the urine and the intensity of immunostaining was correlated with the stage of the disease. These data strongly suggest that acidic fibroblast growth factor is a potential marker for bladder tumors that may be of use in the noninvasive followup of patients with bladder cancer. We present a simple and reliable enzyme immunoassay for the detection of acidic fibroblast growth factor in voided urine that might be useful to quantitate this marker.

The 37-kDa/67-kDa Laminin Receptor Acts as a Receptor for Infectious Prions and Is Inhibited by Polysulfated Glycanes

BACKGROUND Recently, we showed that the 37-kDa/67-kDa laminin receptor (LRP/LR) acts as the receptor of the cellular prion protein. METHODS For the present study, we investigated the binding of the murine scrapie prion protein (moPrP27-30) to baby hamster kidney (BHK) cells, using the Semliki Forest virus system. RESULTS The enhanced binding of moPrP27-30 to BHK cells expressing moLRP::FLAG was inhibited by the LRP/LR-specific antibody W3, which suggests that LRP/LR acts as a receptor for the scrapie form of the prion protein, PrP(Sc). This finding was confirmed by a parallel study that showed that bovine prions are internalized by human enterocytes via LRP/LR. The heparan sulfate mimetics HM5004 and HM2602 reduced PrP27-30 binding to moLRP-expressing cells to approximately 30% and approximately 20%, respectively, at a concentration of 10 microg/mL, whereas pentosan polysulfate (SP54) and phycarin sulfate (PS3) both reduced the binding to approximately 40% at a concentration of 100 microg/mL. CONCLUSIONS We suggest that the inhibition reported elsewhere of PrP(Sc) synthesis and the incubation times prolonged in rodent models by these sulfated glycans are due to the inhibition of the LRP/LR-dependent binding of prions to the target cells.

Heparan-like molecules induce the repair of skull defects

Heparin-binding growth factors (HBGFs) are known to stimulate bone repair when applied to bone lesions. Nevertheless, successful treatments are obtained with high protein doses since HBGFs are rapidly degraded in situ by multiple proteolytic activities associated with the inflammatory period of tissue healing. Like heparin or heparan sulfates, heparan-like molecules, named carboxymethyl-benzylamide-sulfonated dextrans (CMDBS), are known to potentiate fibroblast growth factor activities by stabilizing them against pH, thermal or proteolytic denaturations, and by enhancing their binding with cell surface receptors. We have postulated that CMDBS stimulate in vivo bone healing by interacting with endogenous HBGFs, spontaneously released in the wounded site. The effect of CMDBS on bone repair was studied in a skull defect model in rats by computer-assisted radio-morphometry and histomorphometry. Single application of CMDBS in a collagen vehicle to skull defects induced a dose-dependent increase in bone defect closure and new bone formation after 35 days. Complete bony bridging occurred in defects treated with 3 micrograms CMDBS, whereas bone formation was not observed in vehicle-treated defects which contained only dense fibrous connective tissue between the defect margins. These results indicate that heparan-like molecules, such as CMDBS, are able to induce bone regeneration of skull defects. This action is possibly mediated by potentiation of endogenous growth factor activities and/or by neutralization of proteolytic activities.

Mitogenic and in vitro angiogenic activity of human recombinant heparin affin regulatory peptide.

We have previously described the purification of a heparin binding growth factor from adult bovine brain named heparin affin regulatory peptide (HARP), which was identical to an uterus derived growth factor named pleiotrophin and to a developmentally regulated neurite promoting factor named heparin-binding growth associated molecule. However, for yet unclear reasons, the mitogenic activity of this purified polypeptide following isolation from animal tissue extracts is a subject of controversy, due to conflicting and irreproducible data when produced by recombinant DNA technologies in E. coli or insect cells. The purified protein was inactive in mitogenic assays but the natural molecule was active in assay of neurite outgrowth. In order to clarify these conflicting results and to obtain a recombinant protein free from other contaminating heparin-binding growth factors, we have cloned human cDNA encoding human HARP, engineered its expression in NIH 3T3 cells and characterised the resulting recombinant polypeptide. Purified recombinant HARP displayed mitogenic activity for capillary endothelial cells with half-maximal stimulation at approximately 1 ng/ml (55 pM) and induced angiogenesis in an in vitro model. Interestingly, while the NH2 terminal sequence of tissue purified HARP was NH2-GKKEKPEKK, the NH2 terminal sequence of the biologically active recombinant protein was NH2-AEAGKKEKPEKK, corresponding to a three amino acid extended form.