Human T Cell Differentiation Negatively Regulates Telomerase Expression Resulting in Reduced Activation-Induced Proliferation and Survival.

Maintenance of telomeres is essential for preserving T cell proliferative responses yet the precise role of telomerase in human T cell differentiation, function, and aging is not fully understood. Here we analyzed human telomerase reverse transcriptase (hTERT) expression and telomerase activity in six T cell subsets from 111 human adults and found that levels of hTERT mRNA and telomerase activity had an ordered decrease from naïve (TN) to central memory (TCM) to effector memory (TEM) cells and were higher in CD4+ than their corresponding CD8+ subsets. This differentiation-related reduction of hTERT mRNA and telomerase activity was preserved after activation. Furthermore, the levels of hTERT mRNA and telomerase activity were positively correlated with the degree of activation-induced proliferation and survival of T cells in vitro. Partial knockdown of hTERT by an anti-sense oligo in naïve CD4+ cells led to a modest but significant reduction of cell proliferation. Finally, we found that activation-induced levels of telomerase activity in CD4+ TN and TCM cells were significantly lower in old than in young subjects. These findings reveal that hTERT/telomerase expression progressively declines during T cell differentiation and age-associated reduction of activation-induced expression of hTERT/telomerase mainly affects naïve CD4+ T cells and suggest that enhancing telomerase activity could be a strategy to improve T cell function in the elderly.

Skeletal muscle dihydropyridine-sensitive calcium channel (CACNA1S) gene mutations in chinese patients with hypokalemic periodic paralysis.

BACKGROUND: Thyrotoxic periodic paralysis (TPP), familial periodic paralysis (FPP), and sporadic periodic paralysis (SPP) are common causes of hypokalemic periodic paralysis and have similar clinical presentations, thus possibly sharing the identical mutations. METHODS: We analyzed the role of the three known CACNA1S gene mutations (R528H, R1239H, and R1239G) in Chinese patients, including two FPP families, 36 TPP patients, 12 SPP patients, and their relatives. Fifty unrelated healthy subjects were also studied. Genomic DNA was prepared from the peripheral blood of all patients, their family members, and healthy subjects. Mutations of the CACNA1S gene were screened using polymerase chain reaction-based restriction analysis. RESULTS: Two FPP families had the R528H point mutation, but with incomplete penetrance occurring more commonly in men than in women. Only one SPP patient had a de novo mutation (R528H). None of the TPP patients had mutations in the three hot spots. CONCLUSION: Patients with FPP have R528H mutations in the CACNA1S gene. Only a few patients with SPP may share similar mutations with FPP. TPP patients do not carry any of the three known gene mutations.

MicroRNA-125b modulates inflammatory chemokine CCL4 expression in immune cells and its reduction causes CCL4 increase with age.

Chemokines play a pivotal role in regulating the immune response through a tightly controlled expression. Elevated levels of inflammatory chemokines commonly occur with aging but the mechanism underlying this age-associated change is not fully understood. Here, we report the role of microRNA-125b (miR-125b) in regulating inflammatory CC chemokine 4 (CCL4) expression in human immune cells and its altered expression with aging. We first analyzed the mRNA level of CCL4 in eight different types of immune cells including CD4 and CD8 T-cell subsets (naïve, central and effector memory), B cells and monocytes in blood from both young (≤42 years) and old (≥70 years) adults. We observed that monocytes and naïve CD8 T cells expressed higher levels of CCL4 and exhibited an age-related increase in CCL4. We then found the level of miR-125b was inversely correlated with the level of CCL4 in these cells, and the level of miR-125b was reduced in monocytes and naïve CD8 T cells of the old compared to the young adults. Knock-down of miR-125b by shRNA in monocytes and naïve CD8 T cells led to an increase of CCL4 protein, whereas enhanced miR-125b expression by transfection in naïve CD8 T cells resulted in a reduction of the CCL4 mRNA and protein in response to stimulation. Finally, we demonstrated that miR-125b action requires the 'seed' sequence in 3'UTR of CCL4. Together these findings demonstrated that miR-125b is a negative regulator of CCL4 and its reduction is partially responsible for the age-related increase of CCL4.

Intrafamilial phenotype variability in patients with Gitelman syndrome having the same mutations in their thiazide-sensitive sodium/chloride cotransporter.

BACKGROUND: Gitelman syndrome (GS) most often results from mutations in the thiazide-sensitive sodium chloride cotransporter (NCC). Although the severity of symptoms may vary in patients who have the same mutations, a markedly different clinical presentation in family members with identical mutations is truly rare. METHODS: Five patients (3 women and 2 men) belonging to 2 unrelated Chinese families were investigated. All had chronic hypokalemia, renal potassium (K+) wasting, metabolic alkalosis, and normal blood pressure. Direct sequencing of both the NCC and CLCNKB genes were performed. RESULTS: The probands in each family were men. They had very severe hypokalemia and were symptomatic with episodes of paralysis. They had normal plasma magnesium concentrations, normal calcium excretion rates, and impaired maximal urine concentrating ability. In contrast, female family members were asymptomatic. They had laboratory findings typical of GS--less severe hypokalemia, hypomagnesemia, hypocalciuria, and intact maximal renal concentrating ability. Nevertheless, all patients had the same novel pair of NCC mutations and no mutations detected in CLCNKB. CONCLUSION: Differences in sex may help explain the different clinical presentations in these 2 Chinese families with novel NCC mutations. Hypomagnesemia and hypocalciuria are not always present in patients with GS.

Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals.

Renal epithelial cells release ATP constitutively under basal conditions and release higher quantities of purine nucleotide in response to stimuli. ATP filtered at the glomerulus, secreted by epithelial cells along the nephron, and released serosally by macula densa cells for feedback signaling to afferent arterioles within the glomerulus has important physiological signaling roles within kidneys. In autosomal recessive polycystic kidney disease (ARPKD) mice and humans, collecting duct epithelial cells lack an apical central cilium or express dysfunctional proteins within that monocilium. Collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpk ( Tg737 ) ) mouse model of ARPKD lack a well-formed apical central cilium, thought to be a sensory organelle. We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation. Constitutive ATP release under basal conditions was low and not different in mutant versus rescued monolayers. However, genetically rescued principal cell monolayers released ATP three- to fivefold more robustly in response to ionomycin. Principal cell monolayers with fully formed apical monocilia responded three- to fivefold greater to hypotonicity than mutant monolayers lacking monocilia. In support of the idea that monocilia are sensory organelles, intentionally harsh pipetting of medium directly onto the center of the monolayer induced ATP release in genetically rescued monolayers that possessed apical monocilia. Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia. Our data also show that an increase in cytosolic free Ca(2+) primes the ATP pool that is released in response to mechanical stimuli. It also appears that hypotonic cell swelling and mechanical pipetting stimuli trigger release of a common ATP pool. Cilium-competent monolayers responded to flow with an increase in cell Ca(2+) derived from both extracellular and intracellular stores. This flow-induced Ca(2+) signal was less robust in cilium-deficient monolayers. Flow-induced Ca(2+) signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca(2+). Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na(+)) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases.

Transactivation of hsp70-1/2 in geldanamycin-treated human non-small cell lung cancer H460 cells: involvement of intracellular calcium and protein kinase C.

Geldanamycin is an antitumor drug that binds HSP90 and induces a wide range of heat shock proteins, including HSP70s. In this study we report that the induction of HSP70s is dose-dependent in geldanamycin-treated human non-small cell lung cancer H460 cells. Analysis of the induction of HSP70s specific isoform using LC-ESI-MS/MS analysis and Northern blotting showed that HSP70-1/2 are the major inducible forms under geldanamycin treatment. Transactivation of hsp70-1/2 was determined by electrophoretic mobility-shift assay using heat shock element (HSE) as a probe. The signaling pathway mediators involved in hsp70-1/2 transactivation were screened by the kinase inhibitor scanning technique. Pretreatment with serine/threonine protein kinase inhibitors H7 or H8 blocked geldanamycin-induced HSP70-1/2, whereas protein kinase A inhibitor HA1004, protein kinase G inhibitor KT5823, and myosin light chain kinase inhibitor ML-7 had no effect. Furthermore, the protein kinase C (PKC)-specific inhibitor Ro-31-8425 and the Ca2+-dependent PKC inhibitor Gö-6976 diminished geldanamycin-induced HSP70-1/2, suggesting an involvement of the PKC in the process. In addition, geldanamycin treatment causes a transient increase of intracellular Ca2+. Chelating intracellular Ca2+ with BAPTA-AM or depletion of intracellular Ca2+ store with A23187 or thapsigargin significantly decreased geldanamycin-transactivated HSP70-1/2 expression. Taken together, our results demonstrate that geldanamycin-induced specific HSP70-1/2 isoforms expression in H460 cells through signaling pathway mediated by Ca2+ and PKC.

Extracellular zinc and ATP-gated P2X receptor calcium entry channels: New zinc receptors as physiological sensors and therapeutic targets.

In this review, we focus on two attributes of P2X receptor channel function, one essential and one novel. First, we propose that P2X receptors are extracellular sensors as well as receptors and ion channels. In particular, the large extracellular domain (that comprises 70% of the molecular mass of the receptor channel protein) lends itself to be a cellular sensor. Moreover, its exquisite sensitivity to extracellular pH, ionic strength, and multiple ligands evokes the function of a sensor. Second, we propose that P2X receptors are extracellular zinc receptors as well as receptors for nucleotides. We provide novel data in multiple publications and illustrative data in this invited review to suggest that zinc triggers ATP-independent activation of P2X receptor channel function. In this light, P2X receptors are the cellular site of integration between autocrine and paracrine zinc signaling and autocrine and paracrine purinergic signaling. P2X receptors may sense changes in these ligands as well as in extracellular pH and ionic strength and transduce these sensations via calcium and/or sodium entry and changes in membrane potential.