In Vitro Effects of PTH (1-84) on Human Skeletal Muscle-Derived Satellite Cells.

Parathyroid hormone (PTH) is a hormone secreted by the parathyroid glands. Despite its well-known characterized anabolic and catabolic actions on the skeleton, the in vitro effects of PTH on skeletal muscle cells are limited and generally performed on animal models. The aim of this study was to evaluate the effects of a short impulse of PTH (1-84) on the proliferation and the differentiation of skeletal muscle satellite cells isolated from human biopsies. The cells were exposed for 30 min to different concentrations of PTH (1-84), from 10-6 mol/L to 10-12 mol/L. ELISA was used to assay cAMP and the myosin heavy-chain (MHC) protein. The proliferation was assayed by BrdU and the differentiation by RealTime-qPCR. A statistical analysis was performed by ANOVA followed by Bonferroni's test. No significant variations in cAMP and the proliferation were detected in the isolated cells treated with PTH. On the other hand, 10-7 mol/L PTH on differentiated myotubes has shown significant increases in cAMP (p ≤ 0.05), in the expression of myogenic differentiation genes (p ≤ 0.001), and in the MHC protein (p ≤ 0.01) vs. untreated controls. This work demonstrates for the first time the in vitro effects of PTH (1-84) on human skeletal muscle cells and it opens new fields of investigation in muscle pathophysiology.

Oxidative Stress and Inflammation in Osteoporosis: Molecular Mechanisms Involved and the Relationship with microRNAs.

Osteoporosis is characterized by the alteration of bone homeostasis due to an imbalance between osteoclastic bone resorption and osteoblastic bone formation. Estrogen deficiency causes bone loss and postmenopausal osteoporosis, the pathogenesis of which also involves oxidative stress, inflammatory processes, and the dysregulation of the expression of microRNAs (miRNAs) that control gene expression at post-transcriptional levels. Oxidative stress, due to an increase in reactive oxygen species (ROS), proinflammatory mediators and altered levels of miRNAs enhance osteoclastogenesis and reduce osteoblastogenesis through mechanisms involving the activation of MAPK and transcription factors. The present review summarizes the principal molecular mechanisms involved in the role of ROS and proinflammatory cytokines on osteoporosis. Moreover, it highlights the interplay among altered miRNA levels, oxidative stress, and an inflammatory state. In fact, ROS, by activating the transcriptional factors, can affect miRNA expression, and miRNAs can regulate ROS production and inflammatory processes. Therefore, the present review should help in identifying targets for the development of new therapeutic approaches to osteoporotic treatment and improve the quality of life of patients.

Impact of X-Linked Hypophosphatemia on Muscle Symptoms.

X-linked hypophosphatemia (XLH) is the most common hereditary form of rickets and deficiency of renal tubular phosphate transport in humans. XLH is caused by the inactivation of mutations within the phosphate-regulating endopeptidase homolog X-linked (PHEX) gene and follows an X-dominant transmission. It has an estimated frequency of 1 case per 20,000, and over 300 distinct pathogenic variations have been reported that result in an excess of fibroblast growth factor 23 (FGF23) in the serum. Increased levels of FGF23 lead to renal phosphate loss, decreased serum 1,25-dihydroxyvitamin D, and increased metabolism of 1,25-dihydoxyvitamin D, resulting in hypophosphatemia. Major clinical manifestations include rickets, bone deformities, and growth retardation that develop during childhood, and osteomalacia-related fractures or pseudo-fractures, degenerative osteoarthritis, enthesopathy, dental anomalies, and hearing loss during adulthood, which can affect quality of life. In addition, fatigue is also a common symptom in patients with XLH, who experience decreased motion, muscle weakness, and pain, contributing to altered quality of life. The clinical and biomedical characteristics of XLH are extensively defined in bone tissue since skeletal deformations and mineralization defects are the most evident effects of high FGF23 and low serum phosphate levels. However, despite the muscular symptoms that XLH causes, very few reports are available on the effects of FGF23 and phosphate in muscle tissue. Given the close relationship between bones and skeletal muscles, studying the effects of FGF23 and phosphate on muscle could provide additional opportunities to understand the interactions between these two important compartments of the body. By describing the current literature on XLH and skeletal muscle dysfunctions, the purpose of this review is to highlight future areas of research that could contribute to a better understanding of XLH muscular disability and its management.

In Vitro Non-Genomic Effects of Calcifediol on Human Preosteoblastic Cells.

Several recent studies have demonstrated that the direct precursor of vitamin D3, the calcifediol [25(OH)D3], through the binding to the nuclear vitamin D receptor (VDR), is able to regulate the expression of many genes involved in several cellular processes. Considering that itself may function as a VDR ligand, although with a lower affinity, respect than the active form of vitamin D, we have assumed that 25(OH)D3 by binding the VDR could have a vitamin's D3 activity such as activating non-genomic pathways, and in particular we selected mesenchymal stem cells derived from human adipose tissue (hADMSCs) for the in vitro assessment of the intracellular Ca2+ mobilization in response to 25(OH)D3. Our result reveals the ability of 25(OH)D3 to activate rapid, non-genomic pathways, such as an increase of intracellular Ca2+ levels, similar to what observed with the biologically active form of vitamin D3. hADMSCs loaded with Fluo-4 AM exhibited a rapid and sustained increase in intracellular Ca2+ concentration as a result of exposure to 10-5 M of 25(OH)D3. In this work, we show for the first time the in vitro ability of 25(OH)D3 to induce a rapid increase of intracellular Ca2+ levels in hADMSCs. These findings represent an important step to better understand the non-genomic effects of vitamin D3 and its role in endocrine system.

Available In Vitro Models for Human Satellite Cells from Skeletal Muscle.

Skeletal muscle accounts for almost 40% of the total adult human body mass. This tissue is essential for structural and mechanical functions such as posture, locomotion, and breathing, and it is endowed with an extraordinary ability to adapt to physiological changes associated with growth and physical exercise, as well as tissue damage. Moreover, skeletal muscle is the most age-sensitive tissue in mammals. Due to aging, but also to several diseases, muscle wasting occurs with a loss of muscle mass and functionality, resulting from disuse atrophy and defective muscle regeneration, associated with dysfunction of satellite cells, which are the cells responsible for maintaining and repairing adult muscle. The most established cell lines commonly used to study muscle homeostasis come from rodents, but there is a need to study skeletal muscle using human models, which, due to ethical implications, consist primarily of in vitro culture, which is the only alternative way to vertebrate model organisms. This review will survey in vitro 2D/3D models of human satellite cells to assess skeletal muscle biology for pre-clinical investigations and future directions.

Muscle Physiopathology in Parathyroid Hormone Disorders.

Parathyroid hormone disorders are a group of diseases in which secretion of parathormone (PTH) is impaired. The disorders that result are characterized by signs and symptoms associated with the persistent presence of high blood calcium levels (hypercalcemia) related to hyperparathyroidism (PHPT), or reduced blood calcium levels (hypocalcemia) associated with hypoparathyroidism (HypoPT). In addition to the resulting alteration in bone microarchitecture and mass for both pathologies, patients also report problems with skeletal muscle due to a decrease in muscular strength, muscular dysfunction, and myopathies, which can be responsible for an increased risk of instability and fracture. Although the effect of PTH on bone is well established, and numerous studies suggest that PTH has an effect on skeletal muscle, knowledge about cellular e molecular mechanisms of action on skeletal muscle is very limited. Skeletal muscle is a tissue well known for its structural and mechanical actions and is endowed with an extraordinary ability to adapt to physiological changes. Research in skeletal muscle has increased over the last decade, its importance as an endocrine tissue also emerging, becoming itself a target of numerous substances and hormones. Parathyroid hormone disorders represent a starting point to understand whether PTH may have an effect on skeletal muscle. This review analyzes the basic research data reported to date on PTH and skeletal muscle, highlighting the importance of increasing our knowledge in this field of research.

Study of the Expression and Function of Calcium-Sensing Receptor in Human Skeletal Muscle.

Skeletal muscle has an outstanding capacity for regeneration in response to injuries, but there are disorders in which this process is seriously impaired, such as sarcopenia. Pharmacological treatments to restore muscle trophism are not available, therefore, the identification of suitable therapeutic targets that could be useful for the treatment of skeletal reduced myogenesis is highly desirable. In this in vitro study, we explored the expression and function of the calcium-sensing receptor (CaSR) in human skeletal muscle tissues and their derived satellite cells. The results obtained from analyses with various techniques of gene and protein CaSR expression and of its secondary messengers in response to calcium (Ca2+) and CaSR drugs have demonstrated that this receptor is not present in human skeletal muscle tissues, neither in the established satellite cells, nor during in vitro myogenic differentiation. Taken together, our data suggest that, although CaSR is a very important drug target in physiology and pathology, this receptor probably does not have any physiological role in skeletal muscle in normal conditions.

Analysis of a Preliminary microRNA Expression Signature in a Human Telangiectatic Osteogenic Sarcoma Cancer Cell Line.

Telangiectatic osteosarcoma (TOS) is an aggressive variant of osteosarcoma (OS) with distinctive radiographic, gross, microscopic features, and prognostic implications. Despite several studies on OS, we are still far from understanding the molecular mechanisms of TOS. In recent years, many studies have demonstrated not only that microRNAs (miRNAs) are involved in OS tumorigenesis, development, and metastasis, but also that the presence in high-grade types of OS of cancer stem cells (CSCs) plays an important role in tumor progression. Despite these findings, nothing has been described previously about the expression of miRNAs and the presence of CSCs in human TOS. Therefore, we have isolated/characterized a putative CSC cell line from human TOS (TOS-CSCs) and evaluated the expression levels of several miRNAs in TOS-CSCs using real-time quantitative assays. We show, for the first time, the existence of CSCs in human TOS, highlighting the in vitro establishment of this unique stabilized cell line and an identification of a preliminary expression of the miRNA profile, characteristic of TOS-CSCs. These findings represent an important step in the study of the biology of one of the most aggressive variants of OS and the role of miRNAs in TOS-CSC behavior.

Characterization of Skeletal Muscle Endocrine Control in an In Vitro Model of Myogenesis.

Skeletal muscle has remarkable regenerative abilities regulated by a highly orchestrated process involving the activation of cellular and molecular responses, which are dependent on satellite cells. These cells maintain the stem cell population and provide numerous myogenic cells that proliferate, differentiate, fuse and lead to new myofiber formation for a functional contractile tissue. We have isolated and characterized satellite cells obtained from human biopsies and established an in vitro model of myogenesis, evaluating muscle regeneration, monitoring the dynamic increases of the specific myogenic regulatory factors and the final formation of multinucleated myofibers. As the skeletal muscle is an endocrine tissue able of producing many substances that can act on distant organs, and it can be physiologically modulated by a variety of hormones, we embarked in a project of characterization of muscle cell endocrinology machinery. The expression of a large array of hormone receptors was quantified during the process of myogenesis. The results obtained showed a significant and generalized increase of all the tested hormone receptors along the process of differentiation of human cultured cells from myoblasts to myocytes. Interestingly, also the production of the myokine irisin increased in a parallel manner. These findings point to the human cultured myoblasts as an ideal model to characterize the skeletal muscle endocrine machinery and its hormonal regulation.

Muscle endocrinology and its relation with nutrition.

Recent years have demonstrated clear evidence that skeletal muscle is an active endocrine organ. During contraction of muscle fibers, the skeletal muscle produces and releases, into the blood stream, cytokines and other peptides, called myokines, thanks to which it can both communicate with cells locally within the muscle, in an autocrine and paracrine fashion, or with other distant tissues, exerting its endocrine effects. With the progress of sophisticated technologies, the interest towards the skeletal muscle secretome is rapidly grown and the discovery of new myokines represents a prolific field for the identification of new pharmacological approaches for the management and treatment of many clinical diseases. Considering the importance of the muscle proteome and the cross-talk with other organs, the preservation of a skeletal muscle in good health represents a fundamental aspect in life, especially in ageing. Sarcopenia is the age-dependent loss of skeletal muscle mass and strength, bringing to increases of the risk of adverse outcomes, such as physical disability and poor quality of life, as well as alteration of several hormonal networks. For that reasons, the scientific community has risen its interest to find new interventions to prevent and manage the sarcopenia. Adequate nutrition during ages plays a fundamental role in the health and function of the skeletal muscle and it can represents, alone or in combination with physical exercise, a possible preventive measure against sarcopenia. This review will overview the endocrinology of the skeletal muscle, making a focus on food intake as a strategy for preventing skeletal muscle decay.

Calcium Intake in Bone Health: A Focus on Calcium-Rich Mineral Waters.

Calcium is an essential element that plays numerous biological functions in the human body, of which one of the most important is skeleton mineralization. Bone is a mineralized connective tissue in which calcium represents the major component, conferring bone strength and structure. Proper dietary calcium intake is important for bone development and metabolism, and its requirement can vary throughout life. The mineral composition of drinking water is becoming relevant in the modulation of calcium homeostasis. In fact, calcium present in mineral drinking waters is an important quantitative source of calcium intake. This, together with its excellent bioavailability, contributes to the maintenance of the bone health. This article aims to examine studies that assessed the bioavailability of the calcium contained in calcium-rich mineral waters and their impact on bone health, including original data collected in a recent study in humans.

The effect of strontium chloride on human periodontal ligament stem cells.

The complete repair of periodontal structures remains an exciting challenge that prompts researchers to develop new treatments to restore the periodontium. Recent research has suggested strontium ion to be an attractive candidate to improve osteogenic activity. In this study, we have isolated a clonal finite cell line derived from human periodontal ligament (PDL) in order to assess whether and in which way different doses of SrCl2 (from 0.5 to 500 µg/ml) can influence both the proliferation and the mineralization process, for future application in oral diseases. PDL cells were cloned by dilution plating technique and characterized by FACS. Cell proliferation analysis and mineralization were performed by [3H]-thymidine incorporation and spectrofluorometric assay. Results have evidenced that the higher SrCl2 concentrations tested, from 25 to 500 µg/ml, have increased the proliferation activity after only 24 h of treatment. Interestingly, the same higher concentrations have decreased the mineralization, which was conversely increased by the lower ones, from 0.5 to 10 µg/ml. Our findings suggest the possible use of SrCl2 in appropriate delivery systems that release, at different time points, the specific dose, depending on the biological response that we want to induce on periodontal ligament stem cells, providing a more efficient periodontal regeneration.

Establishment and Characterization of a Human Small Cell Osteosarcoma Cancer Stem Cell Line: A New Possible In Vitro Model for Discovering Small Cell Osteosarcoma Biology.

Osteosarcoma (OSA) is the most common primary malignant bone tumor, usually arising in the long bones of children and young adults. There are different subtypes of OSA, among which we find the conventional OS (also called medullary or central osteosarcoma) which has a high grade of malignancy and an incidence of 80%. There are different subtypes of high grade OS like chondroblastic, fibroblastic, osteoblastic, telangiectatic, and the small cell osteosarcoma (SCO). In this study, for the first time, we have isolated, established, and characterized a cell line of cancer stem cells (CSCs) from a human SCO. First of all, we have established a primary finite cell line of SCO, from which we have isolated the CSCs by the sphere formation assay. We have proved their in vitro mesenchymal and embryonic stem phenotype. Additionally, we have showed their neoplastic phenotype, since the original tumor bulk is a high grade osteosarcoma. This research demonstrates the existence of CSCs also in human primary SCO and highlights the establishment of this particular stabilized cancer stem cell line. This will represent a first step into the study of the biology of these cells to discover new molecular targets molecules for new incisive therapeutic strategies against this highly aggressive OSA.

In Vitro Behavior of Human Adipose Tissue-Derived Stem Cells on Poly(ε-caprolactone) Film for Bone Tissue Engineering Applications.

Bone tissue engineering is an emerging field, representing one of the most exciting challenges for scientists and clinicians. The possibility of combining mesenchymal stem cells and scaffolds to create engineered tissues has brought attention to a large variety of biomaterials in combination with osteoprogenitor cells able to promote and regenerate bone tissue. Human adipose tissue is officially recognized as an easily accessible source of mesenchymal stem cells (AMSCs), a significant factor for use in tissue regenerative medicine. In this study, we analyze the behavior of a clonal finite cell line derived from human adipose tissue seeded on poly(ε-caprolactone) (PCL) film, prepared by solvent casting. PCL polymer is chosen for its good biocompatibility, biodegradability, and mechanical properties. We observe that AMSCs are able to adhere to the biomaterial and remain viable for the entire experimental period. Moreover, we show that the proliferation process and osteogenic activity of AMSCs are maintained on the biofilm, demonstrating that the selected biomaterial ensures cell colonization and the development of an extracellular mineralized matrix. The results of this study highlight that AMSCs and PCL film can be used as a suitable model to support regeneration of new bone for future tissue engineering strategies.

Adipose mesenchymal stem cells in the field of bone tissue engineering.

Bone tissue engineering represents one of the most challenging emergent fields for scientists and clinicians. Current failures of autografts and allografts in many pathological conditions have prompted researchers to find new biomaterials able to promote bone repair or regeneration with specific characteristics of biocompatibility, biodegradability and osteoinductivity. Recent advancements for tissue regeneration in bone defects have occurred by following the diamond concept and combining the use of growth factors and mesenchymal stem cells (MSCs). In particular, a more abundant and easily accessible source of MSCs was recently discovered in adipose tissue. These adipose stem cells (ASCs) can be obtained in large quantities with little donor site morbidity or patient discomfort, in contrast to the invasive and painful isolation of bone marrow MSCs. The osteogenic potential of ASCs on scaffolds has been examined in cell cultures and animal models, with only a few cases reporting the use of ASCs for successful reconstruction or accelerated healing of defects of the skull and jaw in patients. Although these reports extend our limited knowledge concerning the use of ASCs for osseous tissue repair and regeneration, the lack of standardization in applied techniques makes the comparison between studies difficult. Additional clinical trials are needed to assess ASC therapy and address potential ethical and safety concerns, which must be resolved to permit application in regenerative medicine.

Drug delivery using composite scaffolds in the context of bone tissue engineering.

INTRODUCTION: Due to the disadvantages of the current bone autograft and allograft in many clinical condition in which bone regeneration is required in large quantity, engineered biomaterials combined with growth factors, such as bone morphogenetic protein-2 (BMP-2), have been demonstrated to be an effective approach in bone tissue engineering, since they can act both as a scaffold and as a drug delivery system to promote bone repair and regeneration. AREA COVERED: Recent advantages in the field of engineered scaffolds have been obtained from the investigation of composite scaffolds designed by the combination of bioceramics, especially hydroxyapatite (HA), and biodegradable polymers, such as poly (D,L-lactide-co-glycolide) (PLGA) and chitosan, in order to realize osteoconductive structures that can mimic the natural properties of bone tissue. Herein it is demonstrated that the incorporation of BMP-2 into different composite scaffolds, by encapsulation, absorption or entrapment, could be advantageous in terms of osteoinduction for new bone tissue engineered scaffolds as drug delivery systems and some of them should be further analyzed to optimized the drug release for future therapeutic applications. EXPERT OPINION: New design concepts and fabrication techniques represent novel challenges for further investigations about the development of scaffolds as a drug delivery system for bone tissue regeneration.

Role of GSH/GSSG redox couple in osteogenic activity and osteoclastogenic markers of human osteoblast-like SaOS-2 cells.

This study comprised a comprehensive analysis of the glutathione (GSH) redox system during osteogenic differentiation in human osteoblast-like SaOS-2 cells. For the first time, a clear relationship between expression of specific factors involved in bone remodeling and the changes in the GSH/oxidized GSH (GSSG) redox couple induced during the early phases of the differentiation and mineralization process is shown. The findings show that the time course of differentiation is characterized by a decrease in the GSH/GSSG ratio, and this behavior is also related to the expression of osteoclastogenic markers. Maintenance of a high GSH/GSSG ratio due to GSH exposure in the early phase of this process increases mRNA levels of osteogenic differentiation markers and mineralization. Conversely, these events are decreased by a low GSH/GSSG ratio in a reversible manner. Redox regulation of runt-related transcription factor-2 (RUNX-2) activation through phosphorylation is shown. An inverse relationship between RUNX-2 activation and extracellular signal-regulated kinases related to GSH redox potential is observed. The GSH/GSSG redox couple also affects osteoclastogenesis, mainly through osteoprotegerin down-regulation with an increase in the ratio of receptor activator of NF-κB ligand to osteoprotegerin and vice versa. No redox regulation of receptor activator of NF-κB ligand expression was found. These results indicate that the GSH/GSSG redox couple may have a pivotal role in bone remodeling and bone redox-dysregulated diseases. They suggest therapeutic use of compounds that are able to modulate not just the GSH level but the intracellular redox system through the GSH/GSSG redox couple.

Role of N-acetylcysteine and GSH redox system on total and active MMP-2 in intestinal myofibroblasts of Crohn's disease patients.

PURPOSE: Intestinal subepithelial myofibroblasts (ISEMFs)(1) are the predominant source of matrix metalloproteinase-2 (MMP-2) in gut, and a decrease in glutathione/oxidized glutathione (GSH/GSSG) ratio, intracellular redox state index, occurs in the ISEMFs of patients with Crohn's disease (CD). The aim of this study is to demonstrate a relationship between MMP-2 secretion and activation and changes of GSH/GSSG ratio in ISEMFs stimulated or not with tumor necrosis factor alpha (TNFα). METHODS: ISEMFs were isolated from ill and healthy colon mucosa of patients with active CD. Buthionine sulfoximine, GSH synthesis inhibitor, and N-acetylcysteine (NAC), precursor of GSH synthesis, were used to modulate GSH/GSSG ratio. GSH and GSSG were measured by HPLC and MMP-2 by ELISA Kit. RESULTS: In cells, stimulated or not with TNFα, a significant increase in MMP-2 secretion and activation, related to increased oxidative stress, due to low GSH/GSSG ratio, was detected. NAC treatment, increasing this ratio, reduced MMP-2 secretion and exhibited a direct effect on the secreted MMP-2 activity. In NAC-treated and TNFα-stimulated ISEMFs of CD patients' MMP-2 activity were restored to physiological value. The involvement of c-Jun N-terminal kinase pathway on redox regulation of MMP-2 secretion has been demonstrated. CONCLUSION: For the first time, in CD patient ISEMFs, a redox regulation of MMP-2 secretion and activation related to GSH/GSSG ratio and inflammatory state have been demonstrated. This study suggests that compounds able to maintain GSH/GSSG ratio to physiological values can be useful to restore normal MMP-2 levels reducing in CD patient intestine the dysfunction of epithelial barrier.

Oxidative state and IL-6 production in intestinal myofibroblasts of Crohn's disease patients.

BACKGROUND: Intestinal subepithelial myofibroblasts (ISEMFs) produce inflammatory cytokines in response to certain stimuli. In the intestine of patients with Crohn's disease (CD), cytokine synthesis is modified and an increased number of myofibroblasts has been observed. The intracellular redox state influences cytokine production and oxidative stress is present in the intestinal mucosa of CD patients. METHODS: This study was performed in ISEMFs isolated from the colon of patients with active CD and in a myofibroblast cell line derived from human colonic mucosa: 18Co cells. Cellular glutathione (GSH) levels were modulated by treatment with buthionine sulfoximine, an inhibitor of GSH synthesis, or N-acetylcysteine, a GSH precursor. GSH and oxidized glutathione (GSSG) levels were measured by high-performance liquid chromatography (HPLC) methods. Interleukin (IL)-6 production was detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: ISEMFs of CD patients exhibited an increased oxidative state due to a decrease in the GSH/GSSG ratio, which is related to an increase in basal IL-6 production or is stimulated by tumor necrosis factor alpha (TNFα) or bacterial products. This relationship was also confirmed in 18Co cells. Phosphorylation and activation of ERK1/2 and p38 MAPK, which are signaling factors involved in the IL-6 synthesis, were also increased when there is oxidative stress in ISEMFs. CONCLUSIONS: This study shows for the first time in ISEMFs of CD patients an increased production of IL-6 synthesis related to the decrease in the GSH/GSSH ratio, suggesting redox regulation with the involvement of specific kinase activation. The present data shed light on the pathogenesis of inflammatory chronic processes and relapses that occur in this pathology.

Redox regulation of ERK1/2 activation induced by sphingosine 1-phosphate in fibroblasts: involvement of NADPH oxidase and platelet-derived growth factor receptor.

BACKGROUND: Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite synthesized after stimulation with growth factors or cytokines. S1P extracellular effects are mediated through specific Gi-protein coupled receptors (GPCRs). Recently, we demonstrated in NIH3T3 fibroblasts stimulated by platelet-derived growth factor (PDGF) or S1P the NADPH oxidase activation and the H(2)O(2) intracellular level increase trough the Gi protein involvement. METHODS: NIH3T3 fibroblast cell cultures were used. Western blot and quantitative analyses by Chemidoc-Quantity-One software were performed. H(2)O(2) level was assayed by fluorescence spectrophotometric analysis, and cell proliferation by counted manually or ELISA kit. RESULTS: This study demonstrates, in NIH 3T3 fibroblasts, a novel redox regulated mechanism of S1P-induced activation of ERK 1/2 related to NADPH oxidase activity and intracellular H(2)O(2) level increase with PDGF receptor tyrosine kinase involvement through a transactivation mechanism. This event is mediated by S1P(1) and S1P(3) receptors by Gi proteins and can contribute to S1P mitogenic signaling. CONCLUSION: These results can be related to mechanisms of cross-talk previously identified between receptor tyrosine kinase, including PDGFreceptor, and several GPCR ligands. GENERAL SIGNIFICANCE: The redox-sensitive ERK1/2 and PDGFr tyrosine kinase activity could be targets for therapies in diseases in which deregulation of intracellular oxidative status and the consequent alteration of S1P and/or PDGF signaling pathway are involved.