The inhibitor of MyoD Family A (I-MFA) regulates megakaryocyte lineage commitment and terminal differentiation.

Hematopoiesis and lineage commitment are regulated by several conserved cell-intrinsic signaling pathways, including MAPKs and ß-catenin/TCF/LEF. The Inhibitor of MyoD Family A (I-MFA), a transcriptional repressor and tumor suppressor gene, interacts with these pathways and is dysregulated in chronic and acute myeloid leukemias, suggesting it may play a role in development and differentiation during hematopoiesis. To study this, immune cell populations in the bone marrow (BM) and periphery were analyzed in mice lacking Mdfi, encoding I-MFA (I-MFA-/-), and wild type (WT) controls. I-MFA-/- mice had reduced spleen and BM cellularity, with significant hyposplenism, compared to WT mice. In blood, total red blood cells and platelet counts were significantly reduced in I-MFA-/- mice, accompanied by a reduction in megakaryocyte (MK)/erythrocyte progenitor cells and an increase in myeloid progenitors in BM compared to WT mice. The K562 cell line exhibits PMA-induced MK differentiation, and shRNA knockdown of I-MFA resulted in reduced differentiation compared to control, with an increase and prolongation in phospho-JNK and phospho-ERK signaling. Overexpression of I-MFA promoted MK differentiation. These results suggest I-MFA plays a cell-intrinsic role in the response to differentiation signals, an effect that can be explored in the context of hematological cancers or other blood proliferative disorders.

Control of PTH secretion by the TRPC1 ion channel.

Familial hypocalciuric hypercalcemia (FHH) is a genetic condition associated with hypocalciuria, hypercalcemia, and, in some cases, inappropriately high levels of circulating parathyroid hormone (PTH). FHH is associated with inactivating mutations in the gene encoding the Ca2+-sensing receptor (CaSR), a GPCR, and GNA11 encoding G protein subunit α 11 (Gα11), implicating defective GPCR signaling as the root pathophysiology for FHH. However, the downstream mechanism by which CaSR activation inhibits PTH production/secretion is incompletely understood. Here, we show that mice lacking the transient receptor potential canonical channel 1 (TRPC1) develop chronic hypercalcemia, hypocalciuria, and elevated PTH levels, mimicking human FHH. Ex vivo and in vitro studies revealed that TRPC1 serves a necessary and sufficient mediator to suppress PTH secretion from parathyroid glands (PTGs) downstream of CaSR in response to high extracellular Ca2+ concentration. Gα11 physically interacted with both the N- and C-termini of TRPC1 and enhanced CaSR-induced TRPC1 activity in transfected cells. These data identify TRPC1-mediated Ca2+ signaling as an essential component of the cellular apparatus controlling PTH secretion in the PTG downstream of CaSR.

The Sorting Nexin 3 Retromer Pathway Regulates the Cell Surface Localization and Activity of a Wnt-Activated Polycystin Channel Complex.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by inactivating mutations in PKD1 (85%) or PKD2 (15%). The ADPKD proteins encoded by these genes, polycystin-1 (PC1) and polycystin-2 (PC2), form a plasma membrane receptor-ion channel complex. However, the mechanisms controlling the subcellular localization of PC1 and PC2 are poorly understood. Here, we investigated the involvement of the retromer complex, an ancient protein module initially discovered in yeast that regulates the retrieval, sorting, and retrograde transport of membrane receptors. Using yeast two-hybrid, biochemical, and cellular assays, we determined that PC2 binds two isoforms of the retromer-associated protein sorting nexin 3 (SNX3), including a novel isoform that binds PC2 in a direct manner. Knockdown of SNX3 or the core retromer protein VPS35 increased the surface expression of endogenous PC1 and PC2 in vitro and in vivo and increased Wnt-activated PC2-dependent whole-cell currents. These findings indicate that an SNX3-retromer complex regulates the surface expression and function of PC1 and PC2. Molecular targeting of proteins involved in the endosomal sorting of PC1 and PC2 could lead to new therapeutic approaches in ADPKD.

Store-operated calcium entry contributes to abnormal Ca²âº signalling in dystrophic mdx mouse myoblasts.

Sarcolemma damage and activation of various calcium channels are implicated in altered Ca(2+) homeostasis in muscle fibres of both Duchenne muscular dystrophy (DMD) sufferers and in the mdx mouse model of DMD. Previously we have demonstrated that also in mdx myoblasts extracellular nucleotides trigger elevated cytoplasmic Ca(2+) concentrations due to alterations of both ionotropic and metabotropic purinergic receptors. Here we extend these findings to show that the mdx mutation is associated with enhanced store-operated calcium entry (SOCE). Substantially increased rate of SOCE in mdx myoblasts in comparison to that in control cells correlated with significantly elevated STIM1 protein levels. These results reveal that mutation in the dystrophin-encoding Dmd gene may significantly impact cellular calcium response to metabotropic stimulation involving depletion of the intracellular calcium stores followed by activation of the store-operated calcium entry, as early as in undifferentiated myoblasts. These data are in agreement with the increasing number of reports showing that the dystrophic pathology resulting from dystrophin mutations may be developmentally regulated. Moreover, our results showing that aberrant responses to extracellular stimuli may contribute to DMD pathogenesis suggest that treatments inhibiting such responses might alter progression of this lethal disease.

Running performance at high running velocities is impaired but V'O(2max) and peripheral endothelial function are preserved in IL-6⁻/⁻ mice.

It has been reported that IL-6 knockout mice (IL-6⁻/⁻) possess lower endurance capacity than wild type mice (WT), however the underlying mechanism is poorly understood. The aim of the present work was to examine whether reduced endurance running capacity in IL-6⁻/⁻ mice is linked to impaired maximal oxygen uptake (V'O(2max)), decreased glucose tolerance, endothelial dysfunction or other mechanisms. Maximal running velocity during incremental running to exhaustion was significantly lower in IL-6⁻/⁻ mice than in WT mice (13.00±0.97 m·min⁻¹ vs. 16.89±1.15 m·min⁻¹, P<0.02, respectively). Moreover, the time to exhaustion during running at 12 m·min⁻¹ in IL-6⁻/⁻ mice was significantly shorter (P<0.05) than in WT mice. V'O(2max) in IL-6⁻/⁻ (n = 20) amounting to 108.3±2.8 ml·kg⁻¹·min⁻¹ was similar as in WT mice (n = 22) amounting to 113.0±1.8 ml·kg⁻¹·min⁻¹, (P = 0.16). No difference in maximal COX activity between the IL-6⁻/⁻ and WT mice in m. soleus and m. gastrocnemius was found. Moreover, no impairment of peripheral endothelial function or glucose tolerance was found in IL-6⁻/⁻ mice. Surprisingly, plasma lactate concentration during running at 8 m·min⁻¹ as well at maximal running velocity in IL-6⁻/⁻ mice was significantly lower (P<0.01) than in WT mice. Interestingly, IL-6⁻/⁻ mice displayed important adaptive mechanisms including significantly lower oxygen cost of running at a given speed accompanied by lower expression of sarcoplasmic reticulum Ca²âº-ATPase and lower plasma lactate concentrations during running at submaximal and maximal running velocities. In conclusion, impaired endurance running capacity in IL-6⁻/⁻ mice could not be explained by reduced V'O(2max), endothelial dysfunction or impaired muscle oxidative capacity. Therefore, our results indicate that IL-6 cannot be regarded as a major regulator of exercise capacity but rather as a modulator of endurance performance. Furthermore, we identified important compensatory mechanism limiting reduced exercise performance in IL-6⁻/⁻ mice.

Caspase-dependent inhibition of store-operated Ca(2+) entry into apoptosis-committed Jurkat cells.

Activation of T-cells triggers store-operated Ca(2+) entry, which begins a signaling cascade leading to induction of appropriate gene expression and eventually lymphocyte proliferation and differentiation. The simultaneous enhancement of Fas ligand gene expression in activated cells allows the immune response to be limited by committing the activated cells to apoptosis. In apoptotic cells the store-operated calcium entry is significantly inhibited. It has been documented that moderate activation of Fas receptor may cause reversible inhibition of store-operated channels by ceramide released from hydrolyzed sphingomyelin. Here we show that activation of Fas receptor in T-cells results in caspase-dependent decrease of cellular STIM1 and Orai1 protein content. This effect may be responsible for the substantial inhibition of Ca(2+) entry into Jurkat cells undergoing apoptosis. In turn, this inhibition might prevent overloading of cells with calcium and protect them against necrosis.

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

Mutation in dystrophin-encoding gene affects energy metabolism in mouse myoblasts.

Duchenne Muscular Dystrophy is characterized by severe defects in differentiated muscle fibers, including abnormal calcium homeostasis and impaired cellular energy metabolism. Here we demonstrate that myoblasts derived from dystrophic (mdx) mouse exhibit reduced oxygen consumption, increased mitochondrial membrane potential, enhanced reactive oxygen species formation, stimulated glycolysis but unaffected total cellular ATP content. Moreover, reduced amounts of specific subunits of the mitochondrial respiratory complexes and ATP-synthase as well as disorganized mitochondrial network were observed. Both the dystrophic and control myoblasts used were derived from a common inbred mouse strain and the only difference between them is a point mutation in the dystrophin-encoding gene, thus these data indicate that this mutation results in multiple phenotypic alterations demonstrating as early as in undifferentiated myoblasts. This finding sheds new light on the molecular mechanisms of Duchenne Muscular Dystrophy pathogenesis.