Growth Factor Modulation Research Articles

11 INVITED Is mTOR a good target for chemoprevention and what patients could benefit from mTOR inhibitors?

Mechanistic target of rapamycin (mTOR) is a serine–threonine kinase that functions via two multiprotein complexes, namely mTORC1 and mTORC2, each characterized by different binding partners that confer separate functions. mTORC1 function is tightly regulated by PI3-K/Akt and is sensitive to rapamycin. mTORC2 is sensitive to growth factors, not nutrients, and is associated with rapamycin-insensitivity. mTORC1 regulates protein synthesis and cell growth through downstream molecules: 4E-BP1 (also called EIF4E-BP1) and S6K. Also, mTORC2 is thought to modulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases such as Akt and SGK. Recent evidence has suggested that mTORC2 may play an important role in maintenance of normal as well as cancer cells by virtue of its association with ribosomes, which may be involved in metabolic regulation of the cell. Rapamycin (sirolimus) and its analogs known as rapalogues, such as RAD001 (everolimus) and CCI-779 (temsirolimus), suppress mTOR activity through an allosteric mechanism that acts at a distance from the ATP-catalytic binding site, and are considered incomplete inhibitors. Moreover, these compounds suppress mTORC1-mediated S6K activation, thereby blocking a negative feedback loop, leading to activation of mitogenic pathways promoting cell survival and growth. Consequently, mTOR is a suitable target of therapy in cancer treatments. However, neither of these complexes is fully inhibited by the allosteric inhibitor rapamycin or its analogs. In recent years, new pharmacologic agents have been developed which can inhibit these complexes via ATP-binding mechanism, or dual inhibition of the canonical PI3-K/Akt/mTOR signaling pathway. These compounds include WYE-354, KU-003679, PI-103, Torin1, and Torin2, which can target both complexes or serve as a dual inhibitor for PI3-K/mTOR. This investigation describes the mechanism of action of pharmacological agents that effectively target mTORC1 and mTORC2 resulting in suppression of growth, proliferation, and migration of tumor and cancer stem cells.

Growth regulation of the vascular system: an emerging role for adenosine

The importance of metabolic factors in the regulation of angiogenesis is well understood. An increase in metabolic activity leads to a decrease in tissue oxygenation causing tissues to become hypoxic. The hypoxia initiates a variety of signals that stimulate angiogenesis, and the increase in vascularity that follows promotes oxygen delivery to the tissues. When the tissues receive adequate amounts of oxygen, the intermediate effectors return to normal levels, and angiogenesis ceases. An emerging concept is that adenosine released from hypoxic tissues has an important role in driving the angiogenesis. The following feedback control hypothesis is proposed: AMP is dephosphorylated by ecto-5'-nucleotidase, producing adenosine under hypoxic conditions in the extracellular space adjacent to a parenchymal cell (e.g., cardiomyocyte, skeletal muscle fiber, hepatocyte, etc.). Extracellular adenosine activates A(2) receptors, which stimulates the release of vascular endothelial growth factor (VEGF) from the parenchymal cell. VEGF binds to its receptor (VEGF receptor 2) on endothelial cells, stimulating their proliferation and migration. Adenosine can also stimulate endothelial cell proliferation independently of VEGF, which probably involves modulation of other proangiogenic and antiangiogenic growth factors and perhaps an intracellular mechanism. In addition, hemodynamic factors associated with adenosine-induced vasodilation may have a role in the development and remodeling of the vasculature. Once a new capillary network has been established, and the diffusion/perfusion capabilities of the vasculature are sufficient to supply the parenchymal cells with adequate amounts of oxygen, adenosine and VEGF as well as other proangiogenic and antiangiogenic growth factors return to near-normal levels, thus closing the negative feedback loop. The available data indicate that adenosine might be an essential mediator for up to 50-70% of the hypoxia-induced angiogenesis in some situations; however, additional studies in intact animals will be required to fully understand the quantitative importance of adenosine.

Requirement of Epidermal Growth Factor Receptor for Hyperplasia Induced by E5, a High-Risk Human Papillomavirus Oncogene

Multicellular organisms rely on complex networks of signaling cascades for development, homeostasis, and responses to the environment. These networks involve diffusible signaling molecules, their receptors, and a variety of downstream effectors. Alterations in the expression or function of any one of these factors can contribute to disease, including cancer. Many viruses have been implicated in cancer, and some of these modulate cellular signal transduction cascades to carry out their life cycles. High-risk human papillomaviruses (HPVs), the causative agents of most cervical and anogenital cancers, encode three oncogenes. One of these, E5, has been postulated to transform cells in tissue culture by modulating growth factor receptors. In this study, we generate and characterize transgenic mice in which the E5 gene of the most common high-risk HPV, HPV16, is targeted to the basal layer of the stratified squamous epithelium. In these mice, E5 alters the growth and differentiation of stratified epithelia and induces epithelial tumors at a high frequency. Through the analysis of these mice, we show a requirement of the epidermal growth factor receptor for the hyperplastic properties of E5.

C-Src-null mice exhibit defects in normal mammary gland development and ERα signaling

The c-Src tyrosine kinase has been implicated to play an integral role in modulating growth factor receptor, integrin and steroid receptor function. One class of steroid receptors that c-Src modulates is the estrogen receptor alpha (ERalpha). Although there is strong biochemical evidence supporting a role for c-Src in ERalpha signaling, the consequence of this association is unclear at the biological level. To explore the significance of c-Src in ERalpha signaling, we studied the development of various reproductive organs that are dependent on ERalpha in c-Src-deficient mice. We show that the loss of the c-Src tyrosine kinase correlates with defects in ductal development as well as in uterine and ovarian development. Genetic and biochemical analyses of c-Src-deficient mammary epithelial cells also revealed defects in the ability of mammary epithelial cells to activate a number of signaling pathways in response to exogenous estrogen stimulation. Taken together, these studies demonstrate that c-Src plays a role in ERalpha signaling in vivo.

HOXB13 is downregulated in colorectal cancer to confer TCF4-mediated transactivation

Mutations in the Wnt signalling cascade are believed to cause aberrant proliferation of colorectal cells through T-cell factor-4 (TCF4) and its downstream growth-modulating factors. HOXB13 is exclusively expressed in prostate and colorectum. In prostate cancers, HOXB13 negatively regulates β-catenin/TCF4-mediated transactivation and subsequently inhibits cell growth. To study the role of HOXB13 in colorectal tumorigenesis, we evaluated the expression of HOXB13 in 53 colorectal tumours originated from the distal left colon to rectum with their matching normal tissues using quantitative RT–PCR analysis. Expression of HOXB13 is either lost or diminished in 26 out of 42 valid tumours (62%), while expression of TCF4 RNA is not correlated with HOXB13 expression. TCF4 promoter analysis showed that HOXB13 does not regulate TCF4 at the transcriptional level. However, HOXB13 downregulated the expression of TCF4 and its target gene, c-myc, at the protein level and consequently inhibited β-catenin/TCF-mediated signalling. Functionally, forced expression of HOXB13 drove colorectal cancer (CRC) cells into growth suppression. This is the first description of the downregulation of HOXB13 in CRC and its mechanism of action is mediated through the regulation of TCF4 protein stability. Our results suggest that loss of HOXB13 may be an important event for colorectal cell transformation, considering that over 90% of colorectal tumours retain mutations in the APC/β-catenin pathway.

215. Investigating Urokinase Plasminogen Activator Receptor (CD87), as a Potential Target for Non-Viral Gene Transfer

The identification of cell membrane receptors for targeted delivery is important for non-viral gene therapy. The cell surface-associated urokinase-type plasminogen activator (uPA) system plays an important role in tumour cell invasion and metastasis. uPA receptor is a glycosylphosphatidyl inositol linked proteoglycan, over-expressed in malignant tumours compared to normal tissues. We selected two peptide sequences from the amino terminal fragment (ATF), responsible for receptor (uPAR) binding. These peptides, u7 (CLNGGTC) and u11 (VSNKYFSNIHW) are derived from the growth factor module of the ATF.

A novel function for the protein tyrosine phosphatase Shp2 during lung branching morphogenesis

Branching morphogenesis of many organs, including the embryonic lung, is a dynamic process in which growth factor mediated tyrosine kinase receptor activation is required, but must be tightly regulated to direct ramifications of the terminal branches. However, the specific regulators that modulate growth factor signaling downstream of the tyrosine kinase receptor remain to be determined. Herein, we demonstrate for the first time an important function for the intracellular protein tyrosine phosphatase Shp2 in directing embryonic lung epithelial morphogenesis. We show that Shp2 is specifically expressed in embryonic lung epithelial buds, and that loss of function by the suppression of Shp2 mRNA expression results in a 53% reduction in branching morphogenesis. Furthermore, by intra-tracheal microinjection of a catalytically inactive adenoviral Shp2 construct, we provide direct evidence that the catalytic activity of Shp2 is required for proper embryonic lung branch formation. We demonstrate that Shp2 activity is required for FGF10 induced endodermal budding. Furthermore, a loss of Shp2 catalytic activity in the embryonic lung was associated with a reduction in ERK phosphorylation and epithelial cell proliferation. However, epithelial cell differentiation was not affected. Our results show that the protein tyrosine phosphatase Shp2 plays an essential role in modulating growth factor mediated tyrosine kinase receptor activation in early embryonic lung branching morphogenesis.

Heat Shock Protein 90 Stabilization of ErbB2 Expression Is Disrupted by ATP Depletion in Myocytes

Heat shock protein (Hsp) 90 is a ubiquitously expressed chaperone that stabilizes expression of multiple signaling kinases involved in growth regulation, including ErbB2, Raf-1, and Akt. The chaperone activity of Hsp90 requires ATP, which binds with approximately 10-fold lower affinity than ADP. This suggests that Hsp90 may be a physiological ATP sensor, regulating the stability of growth signaling cascades in relation to cellular energy charge. Here we show that lowering ATP concentration by inhibiting glycolysis or mitochondrial respiration in isolated myocytes triggers rapid dissociation of Hsp90 from ErbB2 and degradation of ErbB2 along with other client proteins. The effect of disrupting Hsp90 chaperone activity by ATP depletion was similar to the effect of the pharmacological Hsp90 inhibitor geldanamycin. ATP depletion-induced disruption of Hsp90 chaperone activity was associated with cellular resistance to growth factor activation of intracellular signaling. ErbB2 degradation was also induced by the physiological stress of beta-adrenergic receptor stimulation in electrically stimulated cells. These results support a role for Hsp90 as an ATP sensor that modulates tissue growth factor responsiveness under metabolically stressed conditions and provide a novel mechanism by which cellular responsiveness to growth factor stimulation is modulated by cellular energy charge.

Differential growth factor induction and modulation of human gastric epithelial regeneration

While several autocrine/paracrine growth factors (GFs) can all stimulate epithelial regeneration in experimentally wounded primary gastric cultures, clinical relevance for their non-redundant cooperative actions in human gastric ulcer healing is suggested by the sequential pattern of GF gene induction in vivo. Using new HGE cell lines able to form a coherent monolayer with tight junctions as well as using primary human gastric epithelial cultures, we show that EGF, TGFα, HGF and IGFs accelerate epithelial restitution upon wounding, independently of the TGFβ pathway (as opposed to intestinal cells). However, they differently modulate cell behavior: TGFα exerts strong effects (even more than EGF) on cytoplasmic spreading and non-oriented protruding activity of bordering cells whereas HGF preferentially coordinates single lamella formation, cell elongation and migration into the wound. IGF-I and IGF-II rather induce the alignment of bordering cells and maintain a compact monolayer front. The number of mitotic cells maximally increases with EGF, followed by TGFα and IGF-I,-II. The current study demonstrates that GFs differentially regulate the regeneration of human gastric epithelial cells through specific modulation of cell shape adaptation, migration and proliferation, further stressing that a coordination of GF activities would be necessary for the normal progression of post-wounding epithelial repair.

Autocrine production of prostaglandin F2α enhances phenotypic transformation of normal rat kidney fibroblasts

We have used normal rat kidney (NRK) fibroblasts as an in vitro model system to study cell transformation. These cells obtain a transformed phenotype upon stimulation with growth-modulating factors such as retinoic acid (RA) or transforming growth factor-beta (TGF-beta). Patch-clamp experiments showed that transformation is paralleled by a profound membrane depolarization from around -70 to -20 mV. This depolarization is caused by a compound in the medium conditioned by transformed NRK cells, which enhances intracellular Ca2+ levels and thereby activates Ca2+-dependent Cl- channels. This compound was identified as prostaglandin F2alpha (PGF2alpha) using electrospray ionization mass spectrometry. The active concentration in the medium conditioned by transformed NRK cells as determined using an enzyme immunoassay was 19.7 +/- 2.5 nM (n = 6), compared with 1.5 +/- 0.1 nM (n = 3) conditioned by nontransformed NRK cells. Externally added PGF2alpha was able to trigger NRK cells that had grown to density arrest to restart their proliferation. This proliferation was inhibited when the FP receptor (i.e., natural receptor for PGF2alpha) was blocked by AL-8810. RA-induced phenotypic transformation of NRK cells was partially (approximately 25%) suppressed by AL-8810. Our results demonstrate that PGF2alpha acts as an autocrine enhancer and paracrine inducer of cell transformation and suggest that it may play a crucial role in carcinogenesis in general.

Modulation of angiogenesis in patients with myelodysplastic syndrome

Current investigations have revealed that angiogenesis plays a role in the pathogenesis of high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia, and in the mechanisms of disease progression. Secretion of cytokines and growth factors modulates angiogenesis in the marrow leading to increased vascularity and sustenance of the clonal population. For high-risk MDS patients older than 60 years who are not eligible for aggressive chemotherapy or stem cell transplant, there are few therapeutic options other than supportive treatment. Recent delineation of the pathobiology of MDS has resulted in the development of new agents and treatment modalities that impact on these mechanisms. One of the features of bone marrow pathology is the presence of new vessels, which appear to sustain growth and the hypercellularity of the marrow. Blocking angiogenesis may reduce the microvessel density of the marrow, cellularity, and disease progression. Angiogenesis can be targeted by inhibition of vascular endothelial growth factor (VEGF), which modulates new vessel growth, by the use of antibodies aimed at VEGF and its receptors, as well as receptor tyrosine kinases that block VEGF signaling. Other agents include inhibitors of farnesyl transferase and protein kinase C, which affect upstream modulators of growth factors and their receptor interactions; matrix metalloproteinases, which disrupt matrices and adhesion function promoting vessel growth; and other inhibitors with broader function, such as endostatin, thalidomide, and related analogues

Platelet function in sepsis

Coagulation abnormalities and thrombocytopenia are common in severe sepsis, but sepsis-related alterations in platelet function are ill-defined. The purpose of this study was to elucidate the effect of sepsis on platelet aggregation, adhesiveness, and growth factor release. Agonist-induced platelet aggregation was measured in platelet-rich plasma separated from blood samples collected from 47 critically ill patients with sepsis of recent onset. Expression of platelet adhesion molecules was measured by flow cytometry and the release of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) was measured by ELISA in the supernatant of platelet aggregation. Septic patients had consistently decreased platelet aggregation compared with controls, regardless of the platelet count, thrombin generation, or overt disseminated intravascular coagulation (DIC) status. The severity of sepsis correlated to the platelet aggregation defect. Adhesion molecules, receptor expression (CD42a, CD42b, CD36, CD29, PAR-1), and alpha-granule secretion detected by P-selectin expression remained unchanged but the release of growth factors was differentially regulated with increased VEGF and unchanged PDGF after agonist activation even in uncomplicated sepsis. Sepsis decreases circulating platelets' hemostatic function, maintains adhesion molecule expression and secretion capability, and modulates growth factor production. These results suggest that sepsis alters the hemostatic function of the platelets and increases VEGF release in a thrombin-independent manner.

Pharmacotherapy for diabetic retinopathy

Diabetic retinopathy (DR) is a potentially visually devastating complication of chronic hyperglycemia and other associated systemic abnormalities. Numerous large, prospective, randomized clinical trials have delineated the current standard prevention and treatment protocols including intensive glycemic and blood pressure control and laser photocoagulation for neovascularization and clinically significant macular edema. However, despite standard intervention, vision loss from DR still occurs at an alarming rate. Thus, more recently, researchers have directed their efforts towards better understanding the microscopic changes occurring in DR to develop more effective pharmacologic prevention and treatment strategies. Phase II and III clinical studies involving antivascular endothelial growth factor (VEGF) and protein kinase C (PKC) inhibitors for the management of diabetic macular edema are underway. Researchers recently found elevated pigment endothelium-derived factor (PEDF) associated with active neovascularization, a finding that counteracts prior claims of endogenous anti-angiogenic properties. Other clinical trials are underway to evaluate the efficacy of octreotide, celecoxib, and candesartan on DR. Small clinical studies have suggested beneficial treatment effects for triamcinolone acetonide, interferon alpha-2a, and supplemental oxygen; however, other studies involving losartan, vitamins C and E, and atorvastatin failed to show any benefit. Over the past decade, numerous animal models have led to a more thorough understanding of the early microvascular alterations and later neovascularization and edema observed in DR. These discoveries and subsequent human clinical studies involving direct and indirect growth factor modulation, extracellular matrix alteration, vitreolysis, and alternative DR pathways including dyslipidemia, hypoxia, and sorbitol are reviewed in this manuscript.

Granulin-like peptide in the mid-gut gland of the bivalve mollusk, Patinopecten yessoensis

A cysteine-rich polypeptide, termed CRP1, with a molecular mass of 5829 Da was found to occur in the mid-gut gland of the scallop Patinopecten yessoensis. CRP1 was purified by reverse phase and cation-exchange chromatographies. The amino acid sequence of CRP1 was deduced from its N-terminal amino acid sequence, amino acid composition and the sequence of a partial cDNA, indicating that CRP1 is a 57-amino-acid polypeptide containing 12 cysteine residues with a calculated molecular mass of 5841 Da (5829 Da when oxidized to form six disulfide bridges). A homology search of databases revealed that the deduced amino acid sequence of CRP1 displays significant similarity to those of granulin/epithelins, a family of growth-modulating factors; all cysteine residues in CRP1 are located at the same positions as those conserved characteristically in other known granulin/epithelins. Purified CRP1 inhibited the proliferation of mouse embryo cells. The results suggest that CRP1 functions as a growth-modulating factor in the scallop, and that granulin/epithelin family polypeptides and their precursors play physiologically important roles in invertebrates.

Development of UV-induced squamous cell carcinomas is suppressed in the absence of SPARC.

SPARC (Secreted Protein Acidic and Rich in Cysteine) is a multifunctional glycoprotein belonging to a group of matrix-associated factors that mediate cell-extracellular matrix interactions but have no structural roles. In the present study we investigated the contribution of SPARC to factors that influence the development of skin tumors in response to UV irradiation. A hairless SPARC-null mouse was developed and compared to control SKH1 hairless mice in terms of skin tumor induction and extracellular matrix changes occurring in response to UV-irradiation. Following 23 weeks of exposure to UVB totaling 14.5 J per cm(2), tumor development in the wild-type mice was severe, with an average of over 20 tumors per mouse, many of which were squamous cell carcinomas. Conversely, the SPARC-null mice were strikingly tumor-resistant, developing no squamous cell carcinomas and averaging less than one small papilloma per mouse. SPARC was undetectable immunohistochemically in skin from the non-irradiated control group yet was present in relatively high quantities in the basal and superficial areas of the tumor mass. The SPARC-null mice also exhibited a limited contact hypersensitivity response and were refractory to UV induced immune suppression. In conclusion, SPARC appears to have a crucial role in mediating tumor formation in response to UV irradiation.

Stat1 Controls Postnatal Bone Formation by Regulating Fibroblast Growth Factor Signaling in Osteoblasts

Activation of the signal transducers and activators of transcription (STAT) pathway is important in fibroblast growth factor (FGF) modulation of chondrocyte proliferation and endochondral bone formation during embryogenesis. However, it is not known if the FGF/STAT signaling pathway is important for postnatal bone formation. To examine this, we have characterized a novel skeletal phenotype in Stat1-/- mice in which we find a significant increase in bone mineral density, bone mineral content, and other parameters of bone growth. The data show that osteoblasts derived from Stat1-/- mice have decreased expression of cell cycle inhibitor p21WAF/CIP and FGF receptor 3, a known negative regulator of chondrocyte proliferation. Interestingly, Stat1-/- osteoblasts showed increased expression of FGF18 in vivo and increased responsiveness to FGF18 in vitro. These results suggest a mechanism for the regulation of the osteoblast in which Stat1 functions not only to directly regulate the cell cycle but also to modify the repertoire of FGF receptor expression from a potentially inhibitory receptor, FGFR3 to a stimulatory receptor such as FGFR1 or FGFR2.

Abrogation of heparan sulfate synthesis in Drosophila disrupts the Wingless, Hedgehog and Decapentaplegic signaling pathways.

Studies in Drosophila and vertebrate systems have demonstrated that heparan sulfate proteoglycans (HSPGs) play crucial roles in modulating growth factor signaling. We have isolated mutations in sister of tout velu (sotv), a gene that encodes a co-polymerase that synthesizes HSPG glycosaminoglycan (GAG) chains. Our phenotypic and biochemical analyses reveal that HS levels are dramatically reduced in the absence of Sotv or its partner co-polymerase Tout velu (Ttv), suggesting that both copolymerases are essential for GAG synthesis. Furthermore, we find that mutations in sotv and ttv impair Hh, Wg and Decapentaplegic (Dpp) signaling. This contrasts with previous studies that suggested loss of ttv compromises only Hh signaling. Our results may contribute to understanding the biological basis of hereditary multiple exostoses (HME), a disease associated with bone overgrowth that results from mutations in EXT1 and EXT2, the human orthologs of ttv and sotv.

Activated Fps/Fes partially rescues the in vivo developmental potential of Flk1-deficient vascular progenitor cells

AbstractRelatively little is known about the modulators of the vascular endothelial growth factor A (VEGF-A)/Flk1 signaling cascade. To functionally characterize this pathway, VEGF-A stimulation of endothelial cells was performed. VEGF-A–mediated Flk1 activation resulted in increased translocation of the endogenous Fps/Fes cytoplasmic tyrosine kinase to the plasma membrane and increased tyrosine phosphorylation, suggesting a role for Fps/Fes in VEGF-A/Flk1 signaling events. Addition of a myristoylation consensus sequence to Fps/Fes resulted in VEGF-A–independent membrane localization of Fps/Fes in endothelial cells. Expression of the activated Fps/Fes protein in Flk1-deficient embryonic stem (ES) cells rescued their contribution to the developing vascular endothelium in vivo by using ES cell–derived chimeras. Activated Fps/Fes contributed to this rescue event by restoring the migratory potential to Flk1 null progenitors, which is required for movement of hemangioblasts from the primitive streak region into the yolk sac proper. Activated Fps/Fes in the presence of Flk1 increased the number of hemangioblast colonies in vitro and increased the number of mesodermal progenitors in vivo. These results suggest that Fps/Fes may act synergistically with Flk1 to modulate hemangioblast differentiation into the endothelium. We have also demonstrated that activated Fps/Fes causes hemangioma formation in vivo, independently of Flk1, as a result of increasing vascular progenitor density.

Monitoring intracellular metabolites in neuroblastoma with 1H NMR spectroscopy: effects of growth factor withdrawal and modulation of lipid metabolism

1H NMR spectroscopy has previously been employed to detect and monitor changes in the lipid metabolism of neuroblastoma cells upon cytotoxic treatment. Here, we addressed the question whether altered growth conditions, by presence or absence of serum, would impact on the metabolites detectable with 1H NMR spectroscopy. Chronic serum deprivation of SH‒SY5Y human neuroblastoma cells resulted in a decrease in the intracellular content of several metabolites, in particular total choline. This metabolic effect was paralleled by significant growth inhibition. In addition, we investigated the potential functional origin of intracellular 1H NMR visible lipids in SH‒SY5Y cells. A drop in lipid methylene protons could be observed shortly after serum‒withdrawal. Contrary, removal of lipoproteins from the serum led to a pronounced increase in intracellular lipids, as did inhibition of de novo sterol synthesis by lovastatin. In conclusion, we demonstrate that intracellular total choline in neuroblastoma cells in vitro is highly dependent on the availability of growth factors. Furthermore, we show that 1H NMR visible lipids decrease upon serum‒withdrawal but are accumulated when cholesterol supply is abrogated. The biological and potential clinical implications of these findings are discussed.

Heparan sulfate proteoglycans are increased during skeletal muscle regeneration: requirement of syndecan-3 for successful fiber formation.

Skeletal muscle regeneration is a highly complex and regulated process that involves muscle precursor proliferation and differentiation and probably requires the participation of heparin binding growth factors such as FGFs, HGF and TGFbeta. Heparan sulfate proteoglycans, key components of cell-surfaces and ECM, modulate growth factor activities and influence cell growth and differentiation. Their expression in forming muscle masses during development and in cell culture, suggest their participation in the regulation of myogenesis. In the present study, heparan sulfate proteoglycan expression in skeletal muscle regeneration induced by barium chloride injection was evaluated. Expression of muscle differentiation markers and neuromuscular junction (NMJ) components was characterized. Immunoblots with anti-Delta-heparan sulfate antibody showed that four major species--perlecan, glypican, syndecan-3 and syndecan-4--were transiently up-regulated. The first three were detected at the surface or basement membranes of newly formed myotubes by specific indirect immunofluorescence. Syndecan-3, a satellite cell marker, showed the earliest and most significant increase. Experiments involving myoblast grafting into regenerating muscle showed that C2C12 cell clones, with inhibited syndecan-3 expression resulting from antisense transfection, presented a normal proliferation rate but an impaired capacity to fuse and form skeletal muscle fibers. These data constitute the first in vivo evidence suggesting the requirement of a specific heparan sulfate proteoglycan for successful skeletal muscle regeneration.