Inhibition of extracellular matrix assembly induces the expression of osteogenic markers in skeletal muscle cells by a BMP-2 independent mechanism

<p>Abstract</p> <p>Background</p> <p>The conversion of one cell type into another has been suggested to be, at the molecular level, the consequence of change(s) in the expression level of key developmental genes. Myoblasts have the ability to differentiate either to skeletal muscle or osteogenic lineage depending of external stimuli. Extracellular matrix (ECM) has been shown to be essential for skeletal muscle differentiation, through its direct interaction with myoblasts' cell receptors. We attempt to address if ECM also plays a role in the osteogenic differentiation of skeletal muscle cells.</p> <p>Results</p> <p>Inhibition of proteoglycan sulfation by sodium chlorate in myoblast cultures strongly affects ECM synthesis and deposition and induces the expression of the osteogenic lineage markers alkaline phosphatase (ALP) and osteocalcin in mononuclear cells. Induction of ALP by sodium chlorate does not affect the expression of specific muscle determination transcription factors, such as MyoD and Myf-5, in the same cells. The osteogenic transcription factor Cbfa-1 expression is also unaffected. Induction of ALP is not inhibited by a soluble form of BMP receptor IA. This suggests that the deviation of the myogenic pathway of C2C12 myoblasts into the osteogenic lineage by inhibitors of proteoglycan sulfation is BMP-2 independent. The increase of osteogenic markers expression can be totally prevented by an exogenous ECM. Interestingly, a similar BMP-2-independent ALP activity induction can be observed in myoblasts cultured on an ECM previously synthesized by BMP-2 treated myoblasts. Under <it>in vivo </it>conditions of increased ECM turn-over and deposition, as in the <it>mdx </it>dystrophic muscle and during skeletal muscle regeneration, an induction and relocalization of ALP is observed in a subpopulation of skeletal muscle fibers, whereas in normal skeletal muscle, ALP expression is restricted to blood vessels and some endomysial mononuclear cells.</p> <p>Conclusion</p> <p>These results suggest that signals arising from the ECM induce the expression of osteogenic markers in muscle cells by a mechanism independent of BMP-2 and without affecting the expression of key muscle or osteogenic determination genes. An induction and relocalization of ALP is also observed in <it>mdx </it>and regenerating skeletal muscles, <it>in vivo </it>conditions of increased muscle ECM deposition or turnover.</p

BMP-2 induces osterix expression through upregulation of DLX5 and its phosphorylation by p38

Osterix, a zinc-finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osterix null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that bone morphogenetic protein-2 (BMP-2) induces an increase in Osterix expression, which is mediated through a homeodomain sequence located in the proximal region of the Osterix promoter. Our results demonstrate that induction of Dlx5 by BMP-2 mediates Osterix transcriptional activation. First, BMP-2 induction of Dlx5 precedes the induction of Osterix. Second, Dlx5 binds to the BMP-responsive homeodomain sequences both in vitro and in vivo. Third, Dlx5 overexpression and knock-down assays demonstrate its role in activating Osterix expression in response to BMP-2. Furthermore, we show that Dlx5 is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-34 and Ser-217 are the sites phosphorylated by p38. Phosphorylation at Ser-34/217 increases the transactivation potential of Dlx5. Thus, we propose that BMP activates expression of Osterix through the induction of Dlx5 and its further transcriptional activation by p38-mediated phosphorylation

The Wnt and BMP Families of Signaling Morphogens at the Vertebrate Neuromuscular Junction

The neuromuscular junction has been extensively employed in order to identify crucial determinants of synaptogenesis. At the vertebrate neuromuscular synapse, extracellular matrix and signaling proteins play stimulatory and inhibitory roles on the assembly of functional synapses. Studies in invertebrate species have revealed crucial functions of early morphogens during the assembly and maturation of the neuromuscular junction. Here, we discuss growing evidence addressing the function of Wnt and Bone morphogenetic protein (BMP) signaling pathways at the vertebrate neuromuscular synapse. We focus on the emerging role of Wnt proteins as positive and negative regulators of postsynaptic differentiation. We also address the possible involvement of BMP pathways on motor neuron behavior for the assembly and/or regeneration of the neuromuscular junction

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Morphogens in the wiring of the nervous system

Neuronal function relies on the establishment of proper connections between neurons and their target cells during development. This basic statement involves several cellular processes, such as neuronal differentiation, the polarized outgrowth of axons and dendrites from differentiated neurons, and the pathfinding of axons towards target cells. The subsequent recognition of complementary synaptic partners finally triggers the formation, maturation, and maintenance of functional synapses. Morphogens are secreted signaling molecules commanding tissue patterning and cell identity during early embryonic development. Remarkably, growing evidence over the last years arising from different invertebrate and vertebrate model organisms has shown that, after cell fate has been established, morphogens also control the precise wiring and function in the developing and mature nervous system. Accordingly, dysfunctions of the signaling pathways activated by these molecules contribute to synaptic disassembly and altered function in diseases affecting the nervous system. We consider it timely to bring together cumulative evidence pointing to crucial roles for signaling activated by different morphogens in the establishment of precise contacts between neurons and their synaptic partners. Therefore, this research topic issue combines review and research articles aimed to cover the functional relevance of such morphogens on the different steps involved in synaptic assembly and function. Diverse model systems of physiological or pathological conditions have been included, as well as different cellular, biochemical and molecular approaches. Altogether, they contribute in different and complementary ways to build a holistic view of the roles that early development morphogens play during the assembly, maintenance and/or regeneration of functional synapses

Reheating in small-field inflation on the brane: The Swampland Criteria and observational constraints in light of the PLANCK 2018 results

We study cosmological inflation and its dynamics in the framework of the Randall-Sundrum II brane model. In particular, we analyze in detail four representative small-field inflationary potentials, namely Natural inflation, Hilltop inflation, Higgs-like inflation, and Exponential SUSY inflation, each characterized by two mass scales. We constrain the parameters for which a viable inflationary Universe emerges using the latest PLANCK results. Furthermore, we investigate whether or not those models in brane cosmology are consistent with the recently proposed Swampland Criteria, and give predictions for the duration of reheating as well as for the reheating temperature after inflation. Our results show that (i) the distance conjecture is satisfied, (ii) the de Sitter conjecture and its refined version may be avoided, and (iii) the allowed range for the five-dimensional Planck mass, $M_5$, is found to be between $10^5~\textrm{TeV}$ and $10^{12}~\textrm{TeV}$. Our main findings indicate that non-thermal leptogenesis cannot work within the framework of RS-II brane cosmology, at least for the inflationary potentials considered here.Comment: 57 pages, 13 tables, 16 figures, discussion adde

Characterization of Wnt/β-catenin and BMP/Smad signaling pathways in an in vitro model of amyotrophic lateral sclerosis

Different pathways activated by morphogens of the early embryonic development, such as the Wnt and the Bone Morphogenetic Protein (BMP) ligands, are involved in diverse physiological and pathological conditions of the nervous system, including neurodegeneration. In this work, we have analyzed the endogenous activity of the canonical Wnt/β-catenin and BMP/Smad-dependent pathways in an in vitro model of amyotrophic lateral sclerosis (ALS), given by motor neuron-like NSC34 cells stably expressing wild-type or G93A mutated forms of human Cu/Zn superoxide dismutase-1 (SOD1). As ALS-derived motor neurons, NSC34 cells expressing mutated hSOD1 show a decreased proliferation rate, are more susceptible to oxidation-induced cell death and display Golgi fragmentation. In addition, they display an impaired ability to induce the expression of the motor neuronal marker Hb9 and, consistently, to morphologically differentiate into a motor neuronal phenotype. Regarding signaling, our data show that the transcriptional activity associated to the Wnt/β-catenin pathway is decreased, a finding possibly associated to the cytosolic aggregation of β-catenin. In turn, the BMP-dependent phosphorylation of Smad1 and the transcriptional activation of the BMP/Smad pathway is increased in the pathologic model. Together, these findings suggest that Wnt/β-catenin and the BMP-dependent pathways could play relevant roles in the neurodegeneration of motor neurons in the context of ALS

Intracellular pH regulation in rat round spermatids

Intracellular pH has been shown to be an important physiological parameter in cell cycle control and differentiation, aspects that are central to the spermatogenic process. However, the pH regulatory mechanisms in spermatogenic cells have not been systematically explored. In this work, measuring intracellular pH (pH(i)) with a fluorescent probe (BCECF), membrane potential with a fluorescent lipophilic anion (bisoxonol), and net movement of acid using a pH-stat system, we have found that rat round spermatids regulate pH(i) by means of a V-type H+-ATPase, a HCO3- entry pathway, a Na+/HCO3- dependent transport system, and a putative proton conductive pathway. Rat spermatids do not have functional base extruder transport systems. These pH regulatory characteristics seem specially designed to withstand acid challenges, and can generate sustained alkalinization upon acid exit stimulation