Early Deconditioning of Human Skeletal Muscles and the Effects of a Thigh Cuff Countermeasure.

Muscle deconditioning is a major consequence of a wide range of conditions from spaceflight to a sedentary lifestyle, and occurs as a result of muscle inactivity, leading to a rapid decrease in muscle strength, mass, and oxidative capacity. The early changes that appear in the first days of inactivity must be studied to determine effective methods for the prevention of muscle deconditioning. To evaluate the mechanisms of muscle early changes and the vascular effect of a thigh cuff, a five-day dry immersion (DI) experiment was conducted by the French Space Agency at the MEDES Space Clinic (Rangueil, Toulouse). Eighteen healthy males were recruited and divided into a control group and a thigh cuff group, who wore a thigh cuff at 30 mmHg. All participants underwent five days of DI. Prior to and at the end of the DI, the lower limb maximal strength was measured and muscle biopsies were collected from the vastus lateralis muscle. Five days of DI resulted in muscle deconditioning in both groups. The maximal voluntary isometric contraction of knee extension decreased significantly. The muscle fiber cross-sectional area decreased significantly by 21.8%, and the protein balance seems to be impaired, as shown by the reduced activation of the mTOR pathway. Measurements of skinned muscle fibers supported these results and potential changes in oxidative capacity were highlighted by a decrease in PGC1-α levels. The use of the thigh cuff did not prevent muscle deconditioning or impact muscle function. These results suggest that the major effects of muscle deconditioning occur during the first few days of inactivity, and countermeasures against muscle deconditioning should target this time period. These results are also relevant for the understanding of muscle weakness induced by muscle diseases, aging, and patients in intensive care.

Transcription profiling in the liver of undernourished male rat offspring reveals altered lipid metabolism pathways and predisposition to hepatic steatosis.

Clinical and animal studies have reported an association between low birth weight and the development of nonalcoholic fatty liver disease (NAFLD) in offspring. Using a model of prenatal maternal 70% food restriction diet (FR30) in the rat, we previously showed that maternal undernutrition predisposes offspring to altered lipid metabolism in adipose tissue, especially on a high-fat (HF) diet. Here, using microarray-based expression profiling combined with metabolic, endocrine, biochemical, histological, and lipidomic approaches, we assessed whether FR30 procedure sensitizes adult male offspring to impaired lipid metabolism in the liver. No obvious differences were noted in the concentrations of triglycerides, cholesterol, and bile acids in the liver of 4-mo-old FR30 rats whichever postweaning diet was used. However, several clues suggest that offspring's lipid metabolism and steatosis are modified by maternal undernutrition. First, lipid composition was changed (i.e., higher total saturated fatty acids and lower elaidic acid) in the liver, whereas larger triglyceride droplets were observed in hepatocytes of undernourished rats. Second, FR30 offspring exhibited long-term impact on hepatic gene expression and lipid metabolism pathways on a chow diet. Although the transcriptome profile was globally modified by maternal undernutrition, cholesterol and bile acid biosynthesis pathways appear to be key targets, indicating that FR30 animals were predisposed to impaired hepatic cholesterol metabolism. Third, the FR30 protocol markedly modifies hepatic gene transcription profiles in undernourished offspring in response to postweaning HF. Overall, FR30 offspring may exhibit impaired metabolic flexibility, which does not enable them to properly cope with postweaning nutritional challenges influencing the development of nonalcoholic fatty liver.

Effect of diet in females (F1) from prenatally undernourished mothers on metabolism and liver function in the F2 progeny is sex-specific.

PURPOSE: Poor maternal nutrition sensitises to the development of metabolic diseases and obesity in adulthood over several generations. The prevalence increases when offspring is fed with a high-fat (HF) diet after weaning. This study aims to determine whether such metabolic profiles can be transmitted to the second generation and even aggravated when the mothers were exposed to overnutrition, with attention to potential sex differences. METHODS: Pregnant Wistar rats were subjected to ad libitum (control) or 70% food-restricted diet (FR) during gestation (F0). At weaning, F1 females were allocated to three food protocols: (1) standard diet prior to and throughout gestation and lactation, (2) HF diet prior to and standard diet throughout gestation and lactation, and (3) HF diet prior to and throughout gestation and lactation. F2 offspring was studied between 16 and 32 weeks of age. RESULTS: FR-F2 offspring on standard diet showed normal adiposity and had no significant metabolic alterations in adulthood. Maternal HF diet resulted in sex-specific effects with metabolic disturbances more apparent in control offspring exposed to HF diet during gestation and lactation. Control offspring displayed glucose intolerance associated with insulin resistance in females. Female livers overexpressed lipogenesis genes and those of males the genes involved in lipid oxidation. Gene expression was significantly attenuated in the FR livers. Increased physical activity associated with elevated corticosterone levels was observed in FR females on standard diet and in all females from overnourished mothers. CONCLUSIONS: Maternal undernutrition during gestation (F0) improves the metabolic health of second-generation offspring with more beneficial effects in females.

Nuclear poly(A)-binding protein aggregates misplace a pre-mRNA outside of SC35 speckle causing its abnormal splicing.

A short abnormal polyalanine expansion in the polyadenylate-binding protein nuclear-1 (PABPN1) protein causes oculopharyngeal muscular dystrophy (OPMD). Mutated PABPN1 proteins accumulate as insoluble intranuclear aggregates in muscles of OPMD patients. While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice have been established, the molecular mechanisms which trigger pathological defects in OPMD and the role of aggregates remain to be determined. Using exon array, for the first time we have identified several splicing defects in OPMD. In particular, we have demonstrated a defect in the splicing regulation of the muscle-specific Troponin T3 (TNNT3) mutually exclusive exons 16 and 17 in OPMD samples compared to controls. This splicing defect is directly linked to the SC35 (SRSF2) splicing factor and to the presence of nuclear aggregates. As reported here, PABPN1 aggregates are able to trap TNNT3 pre-mRNA, driving it outside nuclear speckles, leading to an altered SC35-mediated splicing. This results in a decreased calcium sensitivity of muscle fibers, which could in turn plays a role in muscle pathology. We thus report a novel mechanism of alternative splicing deregulation that may play a role in various other diseases with nuclear inclusions or foci containing an RNA binding protein.

Open-access synthetic spike-in mRNA-seq data for cancer gene fusions.

BACKGROUND: Oncogenic fusion genes underlie the mechanism of several common cancers. Next-generation sequencing based RNA-seq analyses have revealed an increasing number of recurrent fusions in a variety of cancers. However, absence of a publicly available gene-fusion focused RNA-seq data impedes comparative assessment and collaborative development of novel gene fusions detection algorithms. We have generated nine synthetic poly-adenylated RNA transcripts that correspond to previously reported oncogenic gene fusions. These synthetic RNAs were spiked at known molarity over a wide range into total RNA prior to construction of next-generation sequencing mRNA libraries to generate RNA-seq data. RESULTS: Leveraging a priori knowledge about replicates and molarity of each synthetic fusion transcript, we demonstrate utility of this dataset to compare multiple gene fusion algorithms' detection ability. In general, more fusions are detected at higher molarity, indicating that our constructs performed as expected. However, systematic detection differences are observed based on molarity or algorithm-specific characteristics. Fusion-sequence specific detection differences indicate that for applications where specific sequences are being investigated, additional constructs may be added to provide quantitative data that is specific for the sequence of interest. CONCLUSIONS: To our knowledge, this is the first publicly available synthetic RNA-seq data that specifically leverages known cancer gene-fusions. The proposed method of designing multiple gene-fusion constructs over a wide range of molarity allows granular performance analyses of multiple fusion-detection algorithms. The community can leverage and augment this publicly available data to further collaborative development of analytical tools and performance assessment frameworks for gene fusions from next-generation sequencing data.

Early movement restriction affects the acquisition of neurodevelopmental reflexes in rat pups.

Childhood is a period of construction of the organism, during which interactions with the environment and regular physical activity are necessary for the maturation of the neuronal networks. An atypical sensorimotor activity during childhood (due to bed-rest or neurodevelopmental disorders) impacts the development of the neuromuscular system. A model of sensorimotor restriction (SMR) developed in rats has shown that casting pups' hind limbs from postnatal day 1 (P1) to P28 induced a severe perturbation of motor behavior, due to muscle weakness as well as disturbances within the central nervous system. In the present study, our objective was to determine whether SMR affects the early postnatal ontogenesis. We explored the neuromuscular development through the determination of the age for achievement of the main neurodevelopmental reflexes, which represent reliable indicators of neurological and behavioral development. We also evaluated the maturation of postural control. Our results demonstrate that SMR induces a delay in the motor development, illustrated by a several days delay in the acquisition of a mature posture and in the acquisition reflexes: hind limb grasping, righting, hind limb placing, cliff avoidance, negative geotaxis. In conclusion, impaired physical activity and low interactions with environment during early development result in altered maturation of the nervous system.

Molecular determinants of skeletal muscle force loss in response to 5 days of dry immersion in human.

BACKGROUND: Astronauts in Earth's orbit experience microgravity, resulting in a decline of skeletal muscle mass and function. On Earth, models simulating microgravity have shown that the extent of the loss in muscle force is greater than the loss in muscle mass. The reasons behind this disproportionate loss of muscle force are still poorly understood. In the present study, we hypothesize that alongside the loss in skeletal muscle mass, modifications in the expression profile of genes encoding critical determinants of resting membrane potential, excitation-contraction coupling and Ca2+ handling contribute to the decline in skeletal muscle force. METHODS: Healthy male volunteers (n = 18) participated in a 5-day dry immersion (DI) study, an Earth-based model of simulated microgravity. Muscle force measurement and MRI analysis of the cross-sectional area of thigh muscles were performed before and after DI. Biopsies of the vastus lateralis skeletal muscle performed before and after DI were used for the determination Ca2+ properties of isolated muscle fibres, molecular and biochemical analyses. RESULTS: The extent of the decline in force, measured as maximal voluntary contraction of knee extensors (-11.1%, P < 0.01) was higher than the decline in muscle mass (-2.5%, P < 0.01). The decline in muscle mass was molecularly supported by a significant repression of the anabolic IGF-1/Akt/mTOR pathway (-19.9% and -40.9% in 4E-BP1 and RPS6 phosphorylation, respectively), a transcriptional downregulation of the autophagy-lysosome pathway and a downregulation in the mRNA levels of myofibrillar protein slow isoforms. At the single fibre level, biochemical and tension-pCa curve analyses showed that the loss in force was independent of fibre type (-11% and -12.3% in slow and fast fibres, respectively) and Ca2+ activation properties. Finally, we showed a significant remodelling in the expression of critical players of resting membrane potential (aquaporin 4: -24.9%, ATP1A2: +50.4%), excitation-contraction coupling (CHRNA1: +75.1%, CACNA2D1: -23.5%, JPH2: -24.2%, TRDN: -15.6%, S100A1: +27.2%), and Ca2+ handling (ATP2A2: -32.5%, CASQ1: -15%, ORAI1: -36.2%, ATP2B1: -19.1%). CONCLUSIONS: These findings provide evidence that a deregulation in the expression profile of critical molecular determinants of resting membrane potential, excitation-contraction coupling, and Ca2+ handling could be involved in the loss of muscle force induced by DI. They also provide the paradigm for the understanding of muscle force loss during prolonged bed rest periods as those encountered in intensive care unit.

Potential regulation of human muscle plasticity by MLC2 post-translational modifications during bed rest and countermeasures.

This study investigated the effects of a 60-day bed rest with or without countermeasures on muscular phenotype and post-translational modifications of the regulatory Myosin Light Chain 2 (MLC2) protein. Soleus biopsies were obtained from female subjects before and after bed rest. Control subjects were assigned only to bed rest (BR), BR+Ex subjects were submitted to combined aerobic and resistive exercises, and BR+Nut to nutritional leucine and valine diet. We determined Myosin Heavy Chains (MHC) and MLC2 composition of muscles using 1D SDS-PAGE. MLC2 phosphorylation was measured on 2D gels and O-N-Acetyl Glucosaminylation (O-GlcNAc) level of MLC2 was determined. Our results showed a slow-to-fast shift of MHC and MLC2 isoforms in BR and BR+Nut while BR+Ex combinations prevented these phenotype changes. After BR, the MLC2 phosphorylation state was increased while the global MLC2 glycosylation level was decreased. Exercises prevented the variations of phosphorylation and glycosylation observed after BR whereas nutrition had no effects. These results suggested an interplay between phosphorylation and glycosylation of MLC2, which might be involved in the development of muscle atrophy and associated changes. These findings of differential responses to exercises and nutrition protocols were discussed with implications for future prescription models to preserve muscle against long-term unloading.

uPAR induces epithelial-mesenchymal transition in hypoxic breast cancer cells.

Hypoxia activates genetic programs that facilitate cell survival; however, in cancer, it may promote invasion and metastasis. In this study, we show that breast cancer cells cultured in 1.0% O(2) demonstrate changes consistent with epithelial-mesenchymal transition (EMT). Snail translocates to the nucleus, and E-cadherin is lost from plasma membranes. Vimentin expression, cell migration, Matrigel invasion, and collagen remodeling are increased. Hypoxia-induced EMT is accompanied by increased expression of the urokinase-type plasminogen activator receptor (uPAR) and activation of cell signaling factors downstream of uPAR, including Akt and Rac1. Glycogen synthase kinase-3beta is phosphorylated, and Snail expression is increased. Hypoxia-induced EMT is blocked by uPAR gene silencing and mimicked by uPAR overexpression in normoxia. Antagonizing Rac1 or phosphatidylinositol 3-kinase also inhibits development of cellular properties associated with EMT in hypoxia. Breast cancer cells implanted on chick chorioallantoic membranes and treated with CoCl(2), to model hypoxia, demonstrate increased dissemination. We conclude that in hypoxia, uPAR activates diverse cell signaling pathways that cooperatively induce EMT and may promote cancer metastasis.

Early movement restriction deteriorates motor function and soleus muscle physiology.

Children with low physical activity and interactions with environment experience atypical sensorimotor development and maturation leading to anatomical and functional disorganization of the sensorimotor circuitry and also to enduring altered motor function. Previous data have shown that postnatal movement restriction in rats results in locomotor disturbances, functional disorganization and hyperexcitability of the hind limb representations in the somatosensory and motor cortices, without apparent brain damage. Due to the reciprocal interplay between the nervous system and muscle, it is difficult to determine whether muscle alteration is the cause or the result of the altered sensorimotor behavior (Canu et al., 2019). In the present paper, our objectives were to evaluate the impact of early movement restriction leading to sensorimotor restriction (SMR) during development on the postural soleus muscle and on sensorimotor performance in rats, and to determine whether changes were reversed when typical activity was resumed. Rats were submitted to SMR by hind limb immobilization for 16 h / day from birth to postnatal day 28 (PND28). In situ isometric contractile properties of soleus muscle, fiber cross sectional area (CSA) and myosin heavy chain content (MHC) were studied at PND28 and PND60. In addition, the motor function was evaluated weekly from PND28 to PND60. At PND28, SMR rats presented a severe atrophy of soleus muscle, a decrease in CSA and a force loss. The muscle maturation appeared delayed, with persistence of neonatal forms of MHC. Changes in kinetic properties were moderate or absent. The Hoffmann reflex provided evidence for spinal hyperreflexia and signs of spasticity. Most changes were reversed at PND60, except muscle atrophy. Functional motor tests that require a good limb coordination, i.e. rotarod and locomotion, showed an enduring alteration related to SMR, even after one month of 'typical' activity. On the other hand, paw withdrawal test and grip test were poorly affected by SMR whereas spontaneous locomotor activity increased over time. Our results support the idea that proprioceptive feedback is at least as important as the amount of motor activity to promote a typical development of motor function. A better knowledge of the interplay between hypoactivity, muscle properties and central motor commands may offer therapeutic perspectives for children suffering from neurodevelopmental disorders.

NR1D1 controls skeletal muscle calcium homeostasis through myoregulin repression.

The sarcoplasmic reticulum (SR) plays an important role in calcium homeostasis. SR calcium mishandling is described in pathological conditions, such as myopathies. Here, we investigated whether the nuclear receptor subfamily 1 group D member (NR1D1, also called REV-ERBα) regulates skeletal muscle SR calcium homeostasis. Our data demonstrate that NR1D1 deficiency in mice impaired sarco/endoplasmic reticulum calcium ATPase-dependent (SERCA-dependent) SR calcium uptake. NR1D1 acts on calcium homeostasis by repressing the SERCA inhibitor myoregulin through direct binding to its promoter. Restoration of myoregulin counteracted the effects of NR1D1 overexpression on SR calcium content. Interestingly, myoblasts from patients with Duchenne muscular dystrophy displayed lower NR1D1 expression, whereas pharmacological NR1D1 activation ameliorated SR calcium homeostasis and improved muscle structure and function in dystrophic mdx/Utr+/- mice. Our findings demonstrate that NR1D1 regulates muscle SR calcium homeostasis, pointing to its therapeutic potential for mitigating myopathy.

Maternal prenatal undernutrition programs adipose tissue gene expression in adult male rat offspring under high-fat diet.

Several studies have shown that maternal undernutrition leading to low birth weight predisposes offspring to the development of metabolic pathologies such as obesity. Using a model of prenatal maternal 70% food restriction diet (FR30) in rat, we evaluated whether postweaning high-fat (HF) diet would amplify the phenotype observed under standard diet. We investigated biological parameters as well as gene expression profile focusing on white adipose tissues (WAT) of adult offspring. FR30 procedure does not worsen the metabolic syndrome features induced by HF diet. However, FR30HF rats displayed catch-up growth to match the body weight of adult control HF animals, suggesting an increase of adiposity while showing hyperleptinemia and a blunted increase of corticosterone. Using quantitative RT-PCR array, we demonstrated that FR30HF rats exhibited leptin and Ob-Rb as well as many peptide precursor and receptor gene expression variations in WAT. We also showed that the expression of genes involved in adipogenesis was modified in FR30HF animals in a depot-specific manner. We observed an opposite variation of STAT3 phosphorylation levels, suggesting that leptin sensitivity is modified in WAT adult FR30 offspring. We demonstrated that 11ß-HSD1, 11ß-HSD2, GR, and MR genes are coexpressed in WAT and that FR30 procedure modifies gene expression levels, especially under HF diet. In particular, level variation of 11ß-HSD2, whose protein expression was detected by Western blotting, may represent a novel mechanism that may affect WAT glucocorticoid sensitivity. Data suggest that maternal undernutrition differently programs the adult offspring WAT gene expression profile that may predispose for altered fat deposition.

Cross-Comparison of Human iPSC Motor Neuron Models of Familial and Sporadic ALS Reveals Early and Convergent Transcriptomic Disease Signatures.

Induced pluripotent stem cell (iPSC)-derived neural cultures from amyotrophic lateral sclerosis (ALS) patients can model disease phenotypes. However, heterogeneity arising from genetic and experimental variability limits their utility, impacting reproducibility and the ability to track cellular origins of pathogenesis. Here, we present methodologies using single-cell RNA sequencing (scRNA-seq) analysis to address these limitations. By repeatedly differentiating and applying scRNA-seq to motor neurons (MNs) from healthy, familial ALS, sporadic ALS, and genome-edited iPSC lines across multiple patients, batches, and platforms, we account for genetic and experimental variability toward identifying unified and reproducible ALS signatures. Combining HOX and developmental gene expression with global clustering, we anatomically classified cells into rostrocaudal, progenitor, and postmitotic identities. By relaxing statistical thresholds, we discovered genes in iPSC-MNs that were concordantly dysregulated in postmortem MNs and yielded predictive ALS markers in other human and mouse models. Our approach thus revealed early, convergent, and MN-resolved signatures of ALS.

Reversibility of epithelial-mesenchymal transition (EMT) induced in breast cancer cells by activation of urokinase receptor-dependent cell signaling.

Hypoxia induces expression of the urokinase receptor (uPAR) and activates uPAR-dependent cell signaling in cancer cells. This process promotes epithelial-mesenchymal transition (EMT). uPAR overexpression in cancer cells also promotes EMT. In this study, we tested whether uPAR may be targeted to reverse cancer cell EMT. When MDA-MB 468 breast cancer cells were cultured in 1% O(2), uPAR expression increased, as anticipated. Cell-cell junctions were disrupted, vimentin expression increased, and E-cadherin was lost from cell surfaces, indicating EMT. Transferring these cells back to 21% O(2) decreased uPAR expression and reversed the signs of EMT. In uPAR-overexpressing MDA-MB 468 cells, EMT was reversed by silencing expression of endogenously produced urokinase-type plasminogen activator (uPA), which is necessary for uPAR-dependent cell signaling, or by targeting uPAR-activated cell signaling factors, including phosphatidylinositol 3-kinase, Src family kinases, and extracellular signal-regulated kinase. MDA-MB 231 breast cancer cells express high levels of uPA and uPAR and demonstrate mesenchymal cell morphology under normoxic culture conditions (21% O(2)). Silencing uPA expression in MDA-MB-231 cells decreased expression of vimentin and Snail, and induced changes in morphology characteristic of epithelial cells. These results demonstrate that uPAR-initiated cell signaling may be targeted to reverse EMT in cancer.

The urokinase receptor promotes cancer metastasis independently of urokinase-type plasminogen activator in mice.

The urokinase receptor (uPAR) promotes metastasis of human malignancies; however, its mechanism of action remains incompletely understood. Established models focus on the ability of uPAR to bind urokinase-type plasminogen activator (uPA) and promote protease activation in the tumor cell microenvironment; however, uPAR also regulates cell signaling and migration by both uPA-dependent and -independent mechanisms in vitro. The significance of uPAR as a cell-signaling receptor in vivo remains unclear. In this study, we expressed either human or mouse uPAR in human embryonic kidney (HEK-293) cells. We selected HEK-293 cells because, unlike most cancer cells, they do not express uPA or uPAR endogenously. Both mouse and human uPAR increased cell adhesion and migration on vitronectin. Rac1 was activated and responsible for the increase in cell migration. HEK-293 cells that did not express uPAR formed palpable tumors in severe combined immunodeficient mice; however, metastases were exceedingly rare. The xenografts contained abundant mouse uPA, produced by infiltrating mouse cells, but no human uPA. Mouse uPA bound only to mouse uPAR and not human uPAR and, thus, could not interact with human uPAR-expressing HEK-293 cells in xenografts. Nevertheless, both mouse and human uPAR significantly increased HEK-293 cell metastasis into the lungs. The activity of human uPAR suggests that uPAR may promote cancer metastasis independent of uPA. Candidate mechanisms include its effects on adhesion, migration, and Rac1 activation.

Maternal perinatal undernutrition programs a "brown-like" phenotype of gonadal white fat in male rat at weaning.

Several studies indicate that maternal undernutrition sensitizes the offspring to the development of metabolic disorders, such as obesity. Using a model of perinatal maternal 50% food-restricted diet (FR50), we recently reported that rat neonates from undernourished mothers exhibit decreased leptin plasma levels associated with alterations of hypothalamic proopiomelanocortin system. The present study aimed at examining the consequences of FR50 on the brain-adipose axis in male rat neonates. Using quantitative RT-PCR array containing 84 obesity-related genes, we demonstrated that most of the genes involved in energy metabolism regulation are expressed in rat gonadal white adipose tissue (WAT) and are sensitive to maternal perinatal undernutrition (MPU). In contrast, hypothalamic gene expression was not substantially affected by MPU. Gene expression of uncoupling protein 1 (UCP1), a marker of brown adipocytes, showed an almost 400-fold stimulation in postnatal day 21 (PND21) FR50 animals, suggesting that their gonadal WAT possesses a brown-like phenotype. This was confirmed by histological and immunoshistochemical procedures, which demonstrated that PND21 FR50 gonadal adipocytes are multilocular, resembling those present in interscapular brown adipose tissue, and exhibit an overexpression of UCP1 and neuropeptide Y (NPY) at the protein level. Control animals contained almost exclusively "classical" unilocular white adipocytes that did not show high UCP1 and NPY labeling. After weaning, FR50 animals exhibited a transient hyperphagia that was associated with the disappearance of brown-like fat pads in PND30 WAT. Our results demonstrate that MPU delays the maturation of gonadal WAT during critical developmental time windows, suggesting that it could have long-term consequences on body weight regulation in the offspring.

Expression of melanocyte-related genes in human breast cancer and its implications.

We report the expression of melanocyte-related genes (MRG) in freshly resected, histopathologically confirmed, human breast cancer specimens and describe experiments illuminating similar observations on a variety of breast cancer cell lines including MDA-MB-435. This finding has implications for research on breast cancer, for clinical investigation of cancer patients presenting with metastases from occult primary tumors and for understanding aberrant differentiation in cancer cells. For example, higher expression of six MRG correlated inversely with propensity for metastatic spread in clones isolated from the human breast cancer cell line MDA-MB-435. Comparisons of MRG expression in cells growing in vitro with those seen in tumors generated by the same lines in vivo showed that the levels of activity of these genes are influenced by the surrounding environment. Also, silencing of expression of the melanocyte-related transcription factor MITF, by transduction of the non-metastatic clone NM2C5 with a construct expressing a specific anti-MITF shRNA, resulted in decreased production of 5 of the melanocyte-related proteins including TYRP1, Pmel 17, MART 1(Melan-A) and TYRP2, but no increase in metastatic capability. Hence MRG expression reproducibly ear-marked, but did not cause, metastatic incompetence. We also report cytogenetic and other data that conflict with the recent suggestion that MDA-MB-435 is of melanocytic origin and are more consistent with its original designation as being of mammary lineage. We conclude that detection of MRG expression profiles in freshly excised breast cancers and in cultured breast cancer cells reflects the operationally important clinical phenomenon of inappropriate gene expression in malignant neoplasms. Concomitantly, we suggest that the evidence we have obtained (i) collectively supports the continued widespread use of the MDA-MB-435 cell line in breast cancer and metastasis research and (ii) advances knowledge of the diversity of aberrant differentiation programs in malignant cells, which is valuable for making accurate diagnoses and treatment decisions in clinical oncology.

Phenotypical transitions and Ca2+ activation properties in human muscle fibers: effects of a 60-day bed rest and countermeasures.

Muscle biopsies were taken from soleus and vastus lateralis before and after a 60-day bed rest (BR) to examine expression changes in the regulatory proteins of the thin filament and in contractile function. Twenty-four women separated in three groups were submitted to BR or a combined protocol of resistance and aerobic exercises during BR or received a supplementation of amino acids during BR. Ca(2+)-tension relationships were established in single skinned fibers identified by their myosin heavy chain and troponin C isoform expressions. Expression patterns of regulatory proteins were analyzed on muscle pieces. For both muscles, BR produced similar decreases in slow and fast fiber diameters but larger decreases in P(0) maximal forces in slow than in fast fibers. Specific forces were decreased in slow soleus and vastus fibers, which displayed a reduction in Ca(2+) affinity. These changes were accompanied by slow-to-fast transitions in regulatory proteins, with troponins C and T appearing as sensitive markers of unloading. Exercises prevented the changes in fiber diameters and forces and counteracted most of the slow-to-fast transitions. The nutrition program had a morphological beneficial effect on slow fibers. However, these fibers still presented decreases in specific P(0) after BR. Phenotypical transitions due to BR were not prevented by amino acids. Finally, in vastus lateralis muscle, BR induced a decrease in O-glycosylation level that was prevented by exercise and attenuated by nutrition. In conclusion, this study has addressed for the first time in women the respective efficiencies of two countermeasures associated with BR on muscle properties and regulatory protein expression.

Maternal perinatal undernutrition alters postnatal development of chromaffin cells in the male rat adrenal medulla.

Numerous data suggest that the development of the sympathoadrenal system is highly sensitive to the perinatal environment. We previously reported that maternal perinatal food restriction by 50% (FR50) altered chromaffin cell (CC) organization and activity in offspring at weaning. This study investigated the effects of FR50 on the postnatal time course of CC functional and structural adaptations. FR50 pups exhibited smaller and more abundant scattered clusters of noradrenergic CCs as early as postnatal day 7 (P7), indicating that morphological changes took place earlier during development. At birth, the adrenaline release was defective in FR50 pups, suggesting that maternal FR50 impaired the non-neurogenic control of catecholamine release. At P4, the catecholamine release in response to insulin-induced hypoglycaemia was also absent in FR50 pups. This was associated with the reduction of adrenal catecholamine contents, indicating that the failure to synthesize catecholamine might lead to impaired secretion. We hypothesized that maternal FR50 accelerated the functional connections between CCs and splanchnic nerve endings, leading to the premature loss of the non-neurogenic response. Acetylcholine-containing synaptic endings seemed more precociously functional in FR50 pups, as suggested by increased levels of acetylcholine esterase activity at P14. At P7, insulin-induced hypoglycaemia caused preferential adrenaline release associated with increased catecholamine contents in both groups. However, the response was accentuated in FR50 pups. At P14, the insulin challenge increased plasma levels of adrenaline in control rats, whereas it markedly enhanced the circulating level of both catecholamines in FR50 pups. We demonstrated that maternal FR50 leads to developmentally impaired noradrenergic CC aggregation and advanced splanchnic neurotransmission maturation associated with altered medulla activity in response to metabolic stress. This might contribute to the long-lasting malprogramming of the adrenal medulla and to the development of chronic adult diseases.

The low-density lipoprotein receptor-related protein regulates cancer cell survival and metastasis development.

Low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional receptor involved in receptor-mediated endocytosis and cell signaling. In this study, we show that LRP-1 is abundantly expressed in severe combined immunodeficient (SCID) mouse xenografts by various human cancer cell lines that express very low or undetectable levels of LRP-1 when cultured in 21% O2 in vitro (standard cell culture conditions). To test whether LRP-1 expression in vivo may be explained by hypoxia in the xenografts, CL16 cells, which are derived from the MDA-MB-435 cell line, were cultured in 1.0% O2. A substantial increase in LRP-1 expression was observed. To test the activity of LRP-1 in cancer progression in vivo, LRP-1 expression was silenced in CL16 cells with short hairpin RNA. These cells formed tumors in SCID mice, in which LRP-1 expression remained silenced. Although LRP-1 gene silencing did not inhibit CL16 cell dissemination from the primary tumors to the lungs, the pulmonary metastases failed to enlarge, suggesting compromised survival or growth at the implantation site. In cell culture experiments, significantly increased cell death was observed when LRP-1-silenced CL16 cells were exposed to CoCl2, which models changes that occur in hypoxia. Furthermore, LRP-1-silenced cells expressed decreased levels of vascular endothelial growth factor in response to 1.0% O2. These results suggest mechanisms by which LRP-1 may facilitate the development and growth of cancer metastases in vivo.