A Pharmacological Investigation of the TMEM16A Currents in Murine Skeletal Myogenic Precursor Cells.

TMEM16A is a Ca2+-activated Cl- channel expressed in various species and tissues. In mammalian skeletal muscle precursors, the activity of these channels is still poorly investigated. Here, we characterized TMEM16A channels and investigated if the pharmacological activation of Piezo1 channels could modulate the TMEM16A currents in mouse myogenic precursors. Whole-cell patch-clamp recordings combined with the pharmacological agents Ani9, T16inh-A01 and Yoda1 were used to characterize TMEM16A-mediated currents and the possible modulatory effect of Piezo1 activity on TMEM16A channels. Western blot analysis was also carried out to confirm the expression of TMEM16A and Piezo1 channel proteins. We found that TMEM16A channels were functionally expressed in fusion-competent mouse myogenic precursors. The pharmacological blockage of TMEM16A inhibited myocyte fusion into myotubes. Moreover, the specific Piezo1 agonist Yoda1 positively regulated TMEM16A currents. The findings demonstrate, for the first time, a sarcolemmal TMEM16A channel activity and its involvement at the early stage of mammalian skeletal muscle differentiation. In addition, the results suggest a possible role of mechanosensitive Piezo1 channels in the modulation of TMEM16A currents.

Dipeptide self-assembly into water-channels and gel biomaterial.

Dipeptides are convenient building blocks for supramolecular gel biomaterials that can be produced on a large scale at low cost and do not persist in the environment. In the case of unprotected sequences, hydrophobicity is a key requirement to enable gelation, with Phe-Phe standing out for its self-assembling ability. Conversely, more hydrophilic sequences such as homochiral dipeptides Phe-Val and Val-Phe neither fibrillate nor gel aqueous buffers and their crystal structures reveal amphipathic layers. In this work, we test emerging rules for the design of self-assembling dipeptides using heterochiral Phe-Val and Val-Phe. Each dipeptide is characterized by 1H- and 13C-NMR, LC-MS, circular dichroism, infrared and Raman spectroscopies, rheology, electron microscopy, and single-crystal X-ray diffraction. In particular, D-Phe-L-Val is the first heterochiral dipeptide to self-assemble into supramolecular water-channels whose cavity is defined by four peptide molecules arranged head-to-tail. This minimalistic sequence is devoid of amyloid character as probed by thioflavin T fluorescence and it displays excellent biocompatibility in vitro. The dataset provided, through comparison with the literature, significantly advances the definition of molecular design rules for minimalistic unprotected dipeptides that self-assemble into water-channels and biocompatible gels, to assist with the future development of supramolecular biomaterials with fine control over nanomorphological features for a variety of applications.

Functional constipation masked as irritable bowel syndrome.

BACKGROUND: Rome IV criteria for functional gastrointestinal disorders state that children suspected of having Irritable Bowel Syndrome (IBS) with Constipation (IBS-C) should be preliminarily treated for constipation. We aimed at verifying if functional constipation may indeed lead to an erroneous diagnosis of IBS with diarrhea (IBS-D) or IBS with mixed pattern of diarrhea and constipation (IBS-M). METHODS: We prospectively enrolled in an unblinded fashion 10 and 16 consecutive children referred to our center who met Rome IV criteria for a diagnosis of IBS-D and IBS-M, respectively. Patients who fulfilled criteria for suspect "occult constipation" were then given a bowel cleaning regimen with Polyethylene glycol 3350, re-evaluated at 2 months and followed up for at least 6 months. Sixteen additional patients with IBS with Constipation (IBS-C) referred in the same period served as control. The endpoints were: 1) a decrease of more than 50% in abdominal pain intensity and frequency scores; and 2) for patients with IBS-D and IBS-M: resolution of diarrhea. RESULTS: The endpoints were met by 8 (80%) and 14 (87%) of the patients with IBS-D and IBS-M, respectively, with decrease of abdominal pain and resolution of "diarrhea". The response was not significantly different from that observed in 15 (93%) of the IBS-C control group. CONCLUSION: Acknowledging the limitations of the small number of patients and of the uncontrolled nature of the study, we suggest that a possibly large number of patients labeled as IBS-D or IBS-M may actually simply present functional constipation and should be managed as such.

A "noisy" electrical stimulation protocol favors muscle regeneration in vitro through release of endogenous ATP.

An in vitro system of electrical stimulation was used to explore whether an innovative "noisy" stimulation protocol derived from human electromyographic recordings (EMGstim) could promote muscle regeneration. EMGstim was delivered to cultured mouse myofibers isolated from Flexor Digitorum Brevis, preserving their satellite cells. In response to EMGstim, immunostaining for the myogenic regulatory factor myogenin, revealed an increased percentage of elongated myogenin-positive cells surrounding the myofibers. Conditioned medium collected from EMGstim-treated cell cultures, promoted satellite cells differentiation in unstimulated myofiber cell cultures, suggesting that extracellular soluble factors could mediate the process. Interestingly, the myogenic effect of EMGstim was mimicked by exogenously applied ATP (0.1 µM), reduced by the ATP diphosphohydrolase apyrase and prevented by blocking endogenous ATP release with carbenoxolone. In conclusion, our results show that "noisy" electrical stimulations favor muscle progenitor cell differentiation most likely via the release of endogenous ATP from contracting myofibres. Our data also suggest that "noisy" stimulation protocols could be potentially more efficient than regular stimulations to promote in vivo muscle regeneration after traumatic injury or in neuropathological diseases.

Ticagrelor and tirofiban in pregnancy and delivery: beyond labels.

Pregnancy-associated acute myocardial infarction is a rare condition usually occurring during the third trimester of pregnancy, and associated with three-to-four-fold higher mortality compared with rates among non-pregnant women of the same age. As in non-pregnant women, in cases of ST elevation myocardial infarction, the most effective treatment is primary percutaneous coronary intervention with stent implantation. Unfortunately, management of these patients could be challenging because little is known about the optimal medical strategy; the potential teratogenic effects of the third generation thienopyridines are not fully established too. In fact current guidelines do not provide enough recommendations about tailoring dual antiplatelet therapy prescription according to ischemic profile of the pregnant patients. Moreover, the bleeding risk class of cesarean delivery/hysterectomy is not stated in current consensus documents. We report the second pregnancy-associated acute myocardial infarction case successfully treated with ticagrelor before and after primary percutaneous coronary intervention with drug eluting stent implantation on left coronary artery, but also the first report on use of bridging antiplatelet therapy with tirofiban during temporary withdrawal of ticagrelor because of a C-section.

Nanotubes and water-channels from self-assembling dipeptides.

Dipeptides are attractive building blocks for biomaterials in light of their inherent biocompatibility, biodegradability, and simplicity of preparation. Since the discovery of diphenylalanine (Phe-Phe) self-assembling ability into nanotubes, research efforts have been devoted towards the identification of other dipeptide sequences capable of forming these interesting nanomorphologies, although design rules towards nanotube formation are still elusive. In this review, we analyze the dipeptide sequences reported thus far for their ability to form nanotubes, which often feature water-filled supramolecular channels as revealed by single-crystal X-ray diffraction, as well as their properties, and their potential biological applications, which span from drug delivery and regenerative medicine, to bioelectronics and bioimaging.

Supramolecular Hydrogels from a Tripeptide and Carbon Nano-Onions for Biological Applications.

Nanocomposite hydrogels have attracted researchers' attention in recent years to achieve superior performances in a variety of materials applications. In this work, we describe the outcome of three different strategies to combine a self-assembling tripeptide and carbon nano-onions (CNOs), through covalent and non-covalent approaches, into supramolecular and nanostructured hydrogels. Importantly, the tripeptide coated the nano-onions and extended their aqueous dispersions' stability by several hours. Furthermore, CNOs could be loaded in the tripeptide hydrogels at the highest level ever reported for nanocarbons, indicating high compatibility between the components. The materials were formed in phosphate-buffered solutions, thus paving the way for biological applications, and were characterized by several spectroscopic, microscopic, thermogravimetric, and rheological techniques. In vitro experiments demonstrated excellent cytocompatibility.

"Time window" effect of Yoda1-evoked Piezo1 channel activity during mouse skeletal muscle differentiation.

AIM: Mechanosensitive Piezo1 ion channels emerged recently as important contributors to various vital functions including modulation of the blood supply to skeletal muscles. The specific Piezo1 channel agonist Yoda1 was shown to regulate the tone of blood vessels similarly to physical exercise. However, the direct role of Piezo1 channels in muscle function has been little studied so far. We therefore investigated the action of Yoda1 on the functional state of skeletal muscle precursors (satellite cells and myotubes) and on adult muscle fibres. METHODS: Immunostaining, electrophysiological intracellular recordings and Ca2+ imaging experiments were performed to localize and assess the effect of the chemical activation of Piezo1 channels with Yoda1, on myogenic precursors, adult myofibres and at the adult neuromuscular junction. RESULTS: Piezo1 channels were detected by immunostaining in satellite cells (SCs) and myotubes as well as in adult myofibres. In the skeletal muscle precursors, Yoda1 treatment stimulated the differentiation and cell fusion rather than the proliferation of SCs. Moreover, in myotubes, Yoda1 induced significant [Ca2+ ]i transients, without detectable [Ca2+ ]i response in adult myofibres. Furthermore, although expression of Piezo1 channels was detected around the muscle endplate region, Yoda1 application did not alter either the nerve-evoked or spontaneous synaptic activity or muscle contractions in adult myofibres. CONCLUSION: Our data indicate that the chemical activation of Piezo1 channels specifically enhances the differentiation of skeletal muscle precursors, suggesting a possible new strategy to promote muscle regeneration.

Nanoscale Assembly of Functional Peptides with Divergent Programming Elements.

Self-assembling peptides are being applied both in the biomedical area and as building blocks in nanotechnology. Their applications are closely linked to their modes of self-assembly, which determine the functional nanostructures that they form. This work brings together two structural elements that direct nanoscale self-association in divergent directions: proline as a ß-breaker and the ß-structure-associated diphenylalanine motif, into a single tripeptide sequence. Amino acid chirality was found to resolve the tension inherent to these conflicting self-assembly instructions. Stereoconfiguration determined the ability of each of the eight possible Pro-Phe-Phe stereoisomers to self-associate into diverse nanostructures, including nanoparticles, nanotapes, or fibrils, which yielded hydrogels with gel-to-sol transition at a physiologically relevant temperature. Three single-crystal structures and all-atom molecular dynamics simulations elucidated the ability of each peptide to establish key interactions to form long-range assemblies (i,e., stacks leading to gelling fibrils), medium-range assemblies (i.e., stacks yielding nanotapes), or short-range assemblies (i.e., dimers or trimers that further associated into nanoparticles). Importantly, diphenylalanine is known to serve as a binding site for pathological amyloids, potentially allowing these heterochiral systems to influence the fibrillization of other biologically relevant peptides. To probe this hypothesis, all eight Pro-Phe-Phe stereoisomers were tested in vitro on the Alzheimer's disease-associated Aß(1-42) peptide. Indeed, one nonfibril-forming stereoisomer effectively inhibited Aß fibrillization through multivalent binding between diphenylalanine motifs. This work thus defined heterochirality as a useful feature to strategically develop future therapeutics to interfere with pathological processes, with the additional value of resistance to protease-mediated degradation and biocompatibility.

BDNF impact on synaptic dynamics: extra or intracellular long-term release differently regulates cultured hippocampal synapses.

Brain Derived Neurotrophic Factor (BDNF) signalling contributes to the formation, maturation and plasticity of Central Nervous System (CNS) synapses. Acute exposure of cultured brain circuits to BDNF leads to up-regulation of glutamatergic neuro-transmission, by the accurate tuning of pre and post synaptic features, leading to structural and functional synaptic changes. Chronic BDNF treatment has been comparatively less investigated, besides it may represent a therapeutic option to obtain rescue of post-injury alterations of synaptic networks. In this study, we used a paradigm of BDNF long-term (4 days) incubation to assess in hippocampal neurons in culture, the ability of such a treatment to alter synapses. By patch clamp recordings we describe the augmented function of excitatory neurotransmission and we further explore by live imaging the presynaptic changes brought about by long-term BDNF. In our study, exogenous long-term BDNF exposure of post-natal neurons did not affect inhibitory neurotransmission. We further compare, by genetic manipulations of cultured neurons and BDNF release, intracellular overexpression of this neurotrophin at the same developmental age. We describe for the first-time differences in synaptic modulation by BDNF with respect to exogenous or intracellular release paradigms. Such a finding holds the potential of influencing the design of future therapeutic strategies.

Heterochirality and Halogenation Control Phe-Phe Hierarchical Assembly.

Diphenylalanine is an amyloidogenic building block that can form a versatile array of supramolecular materials. Its shortcomings, however, include the uncontrolled hierarchical assembly into microtubes of heterogeneous size distribution and well-known cytotoxicity. This study rationalized heterochirality as a successful strategy to address both of these pitfalls and it provided an unprotected heterochiral dipeptide that self-organized into a homogeneous and optically clear hydrogel with excellent ability to sustain fibroblast cell proliferation and viability. Substitution of one l-amino acid with its d-enantiomer preserved the ability of the dipeptide to self-organize into nanotubes, as shown by single-crystal XRD analysis, whereby the pattern of electrostatic and hydrogen bonding interactions of the backbone was unaltered. The effect of heterochirality was manifested in subtle changes in the positioning of the aromatic side chains, which resulted in weaker intermolecular interactions between nanotubes. As a result, d-Phe-l-Phe self-organized into homogeneous nanofibrils with a diameter of 4 nm, corresponding to two layers of peptides around a water channel, and yielded a transparent hydrogel. In contrast with homochiral Phe-Phe stereoisomer, it formed stable hydrogels thermoreversibly. d-Phe-l-Phe displayed no amyloid toxicity in cell cultures with fibroblast cells proliferating in high numbers and viability on this biomaterial, marking it as a preferred substrate over tissue-culture plastic. Halogenation also enabled the tailoring of d-Phe-l-Phe self-organization. Fluorination allowed analogous supramolecular packing as confirmed by XRD, thus nanotube formation, and gave intermediate levels of bundling. In contrast, iodination was the most effective strategy to augment the stability of the resulting hydrogel, although at the expense of optical transparency and biocompatibility. Interestingly, iodine presence hindered the supramolecular packing into nanotubes, resulting instead into amphipathic layers of stacked peptides without the occurrence of halogen bonding. By unravelling fine details to control these materials at the meso- and macro-scale, this study significantly advanced our understanding of these systems.

The patterns of spontaneous Ca2+ signals generated by ventral spinal neurons in vitro show time-dependent refinement.

Embryonic spinal neurons maintained in organotypic slice culture are known to mimic certain maturation-dependent signalling changes. With such a model we investigated, in embryonic mouse spinal segments, the age-dependent spatio-temporal control of intracellular Ca(2+) signalling generated by neuronal populations in ventral circuits and its relation with electrical activity. We used Ca(2+) imaging to monitor areas located within the ventral spinal horn at 1 and 2 weeks of in vitro growth. Primitive patterns of spontaneous neuronal Ca(2+) transients (detected at 1 week) were typically synchronous. Remarkably, such transients originated from widespread propagating waves that became organized into large-scale rhythmic bursts. These activities were associated with the generation of synaptically mediated inward currents under whole-cell patch-clamp. Such patterns disappeared during longer culture of spinal segments: at 2 weeks in culture, only a subset of ventral neurons displayed spontaneous, asynchronous and repetitive Ca(2+) oscillations dissociated from background synaptic activity. We observed that the emergence of oscillations was a restricted phenomenon arising together with the transformation of ventral network electrophysiological bursting into asynchronous synaptic discharges. This change was accompanied by the appearance of discrete calbindin immunoreactivity against an unchanged background of calretinin-positive cells. It is attractive to assume that periodic oscillations of Ca(2+) confer a summative ability to these cells to shape the plasticity of local circuits through different changes (phasic or tonic) in intracellular Ca(2+).

Epidermal Growth Factor - based adhesion substrates elicit myoblast scattering, proliferation, differentiation and promote satellite cell myogenic activation.

The biochemical properties of muscle extracellular matrix are essential for stem cell adhesion, motility, proliferation and myogenic development. Recombinant elastin-like polypeptides are synthetic polypeptides that, besides maintaining some properties of the native protein, can be tailored by fusing bioactive sequences to their C-terminal. Our laboratory synthesized several Human Elastin-Like Polypeptides (HELP) derived from the sequence of human tropoelastin. Here, we developed a novel HELP family member by fusing the elastin-like backbone to the sequence of human Epidermal Growth Factor. We employed this synthetic protein, named HEGF, either alone or in combination with other proteins of the HELP family carrying RGD-integrin binding sites, as adhesion substrate for C2C12 myoblasts and satellite cells primary cultures. Adhesion of myoblasts to HEGF-based substrates induced scattering, decreased adhesion and cytoskeleton assembly; the concomitant presence of the RGD motifs potentiated all these effects. Recombinant substrates induced myoblasts proliferation, differentiation and the development of multinucleated myotubes, thus favoring myoblasts expansion and preserving their myogenic potential. The effects induced by adhesion substrates were inhibited by AG82 (Tyrphostin 25) and herbimycin A, indicating their dependence on the activation of both the EGF receptor and the tyrosine kinase c-src. Finally, HEGF increased the number of muscle stem cells (satellite cells) derived from isolated muscle fibers in culture, thus highlighting its potential as a novel substrate for skeletal muscle regeneration strategies.

H(2)O(2) modulates purinergic-dependent calcium signalling in osteoblast-like cells.

Reactive oxygen species (ROS) have long been considered as toxic by-products of aerobic metabolism and appear involved in the pathogenesis of degenerative diseases. The physiological role of ROS as second messengers in cell signal transduction is, on the other hand, increasingly recognized. Here we investigated the effects of H(2)O(2) and extracellular nucleotides on calcium signalling in four osteoblastic cell lines. In the highly differentiated HOBIT cells, sensitive to nanomolar concentrations of ADP and UTP, millimolar H(2)O(2) induced oscillatory increases of the cytosolic calcium concentration followed by a steady and sustained calcium increase. Long lasting rhythmic calcium activity was induced by micromolar H(2)O(2) doses. The H(2)O(2)-induced calcium signals, due to both release from intracellular stores and influx from the extracellular milieu, were totally prevented by incubating the cells with the P2 receptor antagonist suramin or with the ATP/ADP hydrolyzing enzyme apyrase. In the osteosarcoma SaOS-2 cells micromolar H(2)O(2) failed to evoke calcium signals and millimolar H(2)O(2) induced a slowly developing calcium influx which was unaffected by suramin and apyrase. These cells responded to micromolar concentrations of ATP and ADP, but were largely insensitive to UTP. ROS 17/2.8 osteosarcoma cells were totally insensitive to ATP, ADP and UTP in keeping with the evidence that these cells lack functional purinergic receptors. In these cells, H(2)O(2) up to 1mM did not increase the cytosolic calcium concentration. In ROS/P2Y(2) cells, stably expressing the P2Y(2) receptor, spontaneous calcium oscillations were observed in 38% of the population and nanomolar concentration of extracellular ATP or UTP activated oscillations in quiescent cells. Spontaneous calcium signals were inhibited by suramin and apyrase. In these cells H(2)O(2) induced oscillatory calcium activity that was blocked by suramin and apyrase. The sensitivity of ROS/P2Y(2) cells to UTP decreased significantly in the presence of DTT, which was effective also in inhibiting spontaneous calcium oscillations. On the other hand, the membrane-impermeant thiol oxidant DTNB induced calcium oscillations that were inhibited by incubating the cells with suramin or apyrase. Since peroxide did not increase extracellular ATP in these cell lines, we propose that, in osteoblasts, mild oxidative conditions could activate purinergic signalling through the sensitization of P2Y(2) receptor.

Cadmium-Induced Oxidative Stress Impairs Glycemic Control in Adolescents.

Experimental evidence suggests that cadmium (Cd) boosts oxidative stress that may result in toxicity on the endocrine system also in humans. The aim of this study was to investigate the glycemic control and oxidative stress markers in male adolescents with increased urinary levels of cadmium. We investigated 111 males, aged 12-14 years, living in a polluted area of Sicily and a control age-matched population (n = 60) living 28-45 km far from the polluted site. Malondialdehyde (MDA), total antioxidant activity (TAC), metallothionein-1A (MT-1A) gene expression, insulin resistance by the homeostatic model assessment (HOMA-IR), and urinary cadmium were investigated. Cd levels were significantly higher in adolescents living in the polluted area than in control age-matched subjects. Adolescents with elevated Cd levels had a significant increase in MDA, MT-1A, and HOMA-IR and reduced TAC compared to the control group. A robust correlation was found between urinary cadmium and MT-1A, HOMA-IR, and MDA whereas an inverse correlation was identified between urinary cadmium and TAC. This study indicates that cadmium burden alters glycemic control in adolescents and suggests that oxidative stress plays a key role in cadmium-induced insulin resistance, increasing the risk of developing metabolic disorders.

Myoblast adhesion, proliferation and differentiation on human elastin-like polypeptide (HELP) hydrogels.

BACKGROUND: The biochemical, mechanical and topographic properties of extracellular matrix are crucially involved in determining skeletal muscle cell morphogenesis, proliferation and differentiation. Human elastin-like polypeptides (HELPs) are recombinant biomimetic proteins designed to mimic some properties of the native matrix protein; when employed as myoblast adhesion substrates, they stimulate in vitro myogenesis. Given the influence that the biophysical properties of extracellular matrix have on skeletal muscle cells, the aim of this work was to investigate the effects of HELP hydrogels on myoblasts' viability and functions. METHODS: We recently synthesized a novel polypeptide, HELPc, by fusing the elastin-like backbone to a 41aa sequence present in the α2 chain of type IV collagen, containing two arginyl-glycyl-aspartic acid (RGD) motifs. To obtain hydrogels, the enzymatic cross-linking of the HELPc was accomplished by transglutaminase. Here, we employed both non-cross-linked HELPc glass coatings and cross-linked HELPc hydrogels at different monomer densities, as adhesion substrates for C2C12 cells, used as a myoblast model. RESULTS: By comparing cell adhesion, proliferation and differentiation, we revealed several striking differences. Depending on support rigidity, adhesion to HELPc substrates dictated cell morphology, spreading, focal adhesion formation and cytoskeletal organization. Hydrogels greatly stimulated cell proliferation, particularly in low-serum medium, and partially inhibited myogenic differentiation. CONCLUSIONS: On the whole, the results underline the potential of these genetically engineered polypeptides as a tool for dissecting crucial steps in myogenesis.

Extracellular nucleotides activate Runx2 in the osteoblast-like HOBIT cell line: a possible molecular link between mechanical stress and osteoblasts' response.

Dynamic mechanical loading increases bone density and strength and promotes osteoblast proliferation, differentiation and matrix production, by acting at the gene expression level. Molecular mechanisms through which mechanical forces are conversed into biochemical signalling in bone are still poorly understood. A growing body of evidence point to extracellular nucleotides (i.e., ATP and UTP) as soluble factors released in response to mechanical stimulation in different cell systems. Runx2, a fundamental transcription factor involved in controlling osteoblasts differentiation, has been recently identified as a target of mechanical signals in osteoblastic cells. We tested the hypothesis that these extracellular nucleotides could be able to activate Runx2 in the human osteoblastic HOBIT cell line. We found that ATP and UTP treatments, as well as hypotonic stress, promote a significant stimulation of Runx2 DNA-binding activity via a mechanism involving PKC and distinct mitogen-activated protein kinase cascades. In fact, by using the specific inhibitors SB203580 (specific for p38 MAPK) and PD98059 (specific for ERK-1/2 MAPK), we found that ERK-1/2, but not p38, play a major role in Runx2 activation. On the contrary, another important transcription factor, i.e., Egr-1, that we previously demonstrated being activated by extracellular released nucleotides in this osteoblastic cell line, demonstrated to be susceptible to both ERK-1/2 and p38 kinases. These data suggest a possible differential involvement of these two transcription factors in response to extracellularly released nucleotides. The biological relevance of our data is strengthened by the finding that a target gene of Runx2, i.e., Galectin-3, is up-regulated by ATP stimulation of HOBIT cells with a comparable kinetic of that found for Runx2. Since it is known that osteocytes are the primary mechanosensory cells of the bone, we hypothesize that they may signal mechanical loading to osteoblasts through release of extracellular nucleotides. Altogether, these data suggest a molecular mechanism explaining the purinoreceptors-mediated activation of specific gene expression in osteoblasts and could be of help in setting up new pharmacological strategies for the intervention in bone loss pathologies.

Cross-regulation between Egr-1 and APE/Ref-1 during early response to oxidative stress in the human osteoblastic HOBIT cell line: evidence for an autoregulatory loop.

The Early Growth Response protein (Egr-1) is a C(2)H(2)-zinc finger-containing transcriptional regulator involved in the control of cell proliferation and apoptosis. Its DNA-binding activity is redox regulated in vitro through the oxidation-reduction of Cys residues within its DNA-binding domain. APE/Ref-1 is a DNA-repair enzyme with redox modulating activities on several transcription factors. In this study, by evaluating the effects of different stimuli, we found a similar timing of activation being suggestive for a common and co-linear regulation for the two proteins. Indeed, we show that APE/Ref-1 increases the Egr-1 DNA-binding activity in unstimulated osteoblastic HOBIT cells. H(2)O(2) stimulation induces a strong interaction between Egr-1 and APE/Ref-1 at early times upon activation, as assayed by immunoprecipitation experiments. By using a cell transfection approach, we demonstrated the functional role of this interaction showing that two specific Egr-1 target genes, the PTEN phosphatase and the thymidine kinase (TK) genes promoters, are activated by contransfection of APE/Ref-1. Interestingly, by using a cell transfection approach and Chromatin immunoprecipitation assays, we were able to demonstrate that Egr-1 stimulates the transcriptional activity of APE/Ref-1 gene promoter by a direct interaction with specific DNA-binding site on its promoter. Taken together, our data delineate a new molecular mechanism of Egr-1 activation occurring soon after H(2)O(2) stimulation in osteoblastic cells and suggest a model for a positive loop between APE/Ref-1 and Egr-1 that could explain the early transcriptional activation of APE/Ref-1 gene expression.

In vitro myogenesis induced by human recombinant elastin-like proteins.

Mammalian adult skeletal muscle has a limited ability to regenerate after injury, usage or trauma. A promising strategy for successful regenerative technology is the engineering of bio interfaces that mimic the characteristics of the extracellular matrix. Human elastin-like polypeptides (HELPs) have been synthesized as biomimetic materials that maintain some peculiar properties of the native protein. We developed a novel Human Elastin Like Polypeptide obtained by fusing the elastin-like backbone to a domain present in the α2 chain of type IV collagen, containing two RGD motives. We employed this peptide as adhesion substrate for C2C12 myoblasts and compared its effects to those induced by two other polypeptides of the HELP series. Myoblast adhered to all HELPs coatings, where they assumed morphology and cytoarchitecture that depended on the polypeptide structure. Adhesion to HELPs stimulated at a different extent cell proliferation and differentiation, the expression of Myosin Heavy Chain and the fusion of aligned fibers into multinucleated myotubes. Adhesion substrates significantly altered myotubes stiffness, measured by Atomic Force Microscopy, and differently affected the cells Ca(2+) handling capacity and the maturation of excitation-contraction coupling machinery, evaluated by Ca(2+) imaging. Overall, our findings indicate that the properties of HELP biopolymers can be exploited for dissecting the molecular connections underlying myogenic differentiation and for designing novel substrates for skeletal muscle regeneration.

The functional expression of connexin 43 in articular chondrocytes is increased by interleukin 1beta: evidence for a Ca2+-dependent mechanism.

Cell-to-cell interactions and gap junctions-dependent communication are crucially involved in chondrogenic differentiation, while in adult articular cartilage direct intercellular communication occurs mainly among chondrocytes facing the outer cartilage layer. Chondrocytes extracted from adult articular cartilage and grown in primary culture express connexin 43 and form functional gap junctions capable of sustaining the propagation of intercellular Ca2+ waves. Degradation of articular cartilage is a characteristic feature of arthritic diseases and is associated to increased levels of interleukin-1 (IL-1) in the synovial fluid. We have examined the effects of IL-1 on gap junctional communication in cultured rabbit articular chondrocytes. Incubation with IL-1 potentiated the transmission of intercellular Ca2+ waves and the intercellular transfer of Lucifer yellow. The stimulatory effect was accompanied by a dose-dependent increase in the expression of connexin 43 and by an enhanced connexin 43 immunostaining at sites of cell-to-cell contact. IL-1 stimulation induced a dose-dependent increase of cytosolic Ca2+ and activates protein tyrosine phosphorylation. IL-1-dependent up-regulation of connexin 43 could be prevented by intracellular Ca2+ chelation, but not by inhibitors of protein tyrosine kinases, suggesting a crucial role of cytosolic Ca2+ in regulating the expression of connexin 43. IL-1 is one of the most potent cytokines that promotes cartilage catabolism: its modulation of intercellular communication represents a novel mechanism by which proinflammatory mediators regulate the activity of cartilage cells.