Calcium and Vitamin D increase mRNA levels for the growth control hIK1 channel in human epidermal keratinocytes but functional channels are not observed

BACKGROUND: Intermediate-conductance, calcium-activated potassium channels (IKs) modulate proliferation and differentiation in mesodermal cells by enhancing calcium influx, and they contribute to the physiology of fluid movement in certain epithelia. Previous reports suggest that IK channels stimulate proliferative growth in a keratinocyte cell line; however, because these channels indirectly promote calcium influx, a critically unique component of the keratinocyte differentiation program, an alternative hypothesis is that they would be anti-proliferative and pro-differentiating. This study addresses these hypotheses. METHODS: Real-time PCR, patch clamp electrophysiology, and proliferation assays were used to determine if human IK1 (hIK1) expression and function are correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes, and skin biopsies grown in explant culture. RESULTS: hIK1 mRNA expression in human keratinocytes and skin was increased in response to anti-proliferative/pro-differentiating stimuli (elevated calcium and Vitamin D). Correspondingly, the hIK1 agonist 1-EBIO inhibited keratinocyte proliferation suggesting that the channel could be anti-proliferative and pro-differentiating. However, this proliferative inhibition by 1-EBIO was not reversed by a panel of hIK1 blockers, calling into question the mechanism of 1-EBIO action. Subsequent patch clamp electrophysiological analysis failed to detect hIK1 channel currents in keratinocytes, even those expressing substantial hIK1 mRNA in response to calcium and Vitamin D induced differentiation. Identical electrophysiological recording conditions were then used to observe robust IK1 currents in fibroblasts which express IK1 mRNA levels comparable to those of keratinocytes. Thus, the absence of observable hIK1 currents in keratinocytes was not a function of the electrophysiological techniques. CONCLUSION: Human keratinocyte differentiation is stimulated by calcium mobilization and influx, and differentiation stimuli coordinately upregulate mRNA levels of the calcium-activated hIK1 channel. This upregulation is paradoxical in that functional hIK1 channels are not observed in cultured keratinocytes. It appears, therefore, that hIK1 does not contribute to the functional electrophysiology of primary human keratinocytes, nor intact human skin. Further, the results indicate caution is required when interpreting experiments utilizing pharmacological hIK1 modulators in human keratinocytes

Renal blood flow using arterial spin labeling (ASL) MRI: experimental protocol and principles

A noninvasive, robust, and reproducible method to measure renal perfusion is important to understand the physiology of kidney. Arterial spin labeling (ASL) MRI technique labels the endogenous blood water as freely diffusible tracers to measure perfusion quantitatively without relying on exogenous contrast agent. Therefore, it alleviates the safety concern involving gadolinium chelates. To obtain quantitative tissue perfusion information is particularly relevant for multisite and longitudinal imaging of living subjects.This chapter is based upon work from the PARENCHIMA COST Action, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This experimental protocol chapter is complemented by two separate chapters describing the basic concept and data analysis

Quantitative assay for the detection of the V617F variant in the Janus kinase 2 (JAK2) gene using the Luminex xMAP technology

<p>Abstract</p> <p>Background</p> <p>The availability of clinically valid biomarkers contribute to improve the diagnosis and clinical management of diseases. A valine-to-phenylalanine substitution at position 617 (V617F) in the Janus kinase 2 (JAK2) gene has been recently associated with key signaling abnormalities in the transduction of haemopoietic growth-factor receptors and is now considered as a useful clinical marker of myeloproliferative neoplasms. Several methods have recently been reported to detect the JAK2 V617F point mutation and show variable sensitivity.</p> <p>Methods</p> <p>Using the Luminex xMAP technology, we developed a quantitative assay to detect the JAK2V617F variant. The method was based on polymerase chain reaction (PCR) followed by hybridization to specific probes coupled with internally dyed microspheres. The assay comprises 3 steps: genomic DNA extraction, end point PCR reaction, direct hybridization of PCR fragments and quantification. It has been tested with different sources of nucleic acid.</p> <p>Results</p> <p>Applied to whole blood samples, this quantitative assay showed a limit of detection of 2%. A highly sensitive allele-specific primer extension reaction performed in parallel allowed to validate the results and to identify the specimens with values below 2%.</p> <p>Conclusion</p> <p>Direct hybridization assay using the Luminex xMAP technology allows sensitive quantification of JAK2V617F from blood spots. It is simple and can be easily performed in a clinical setting.</p

The Induction of APC with a Distinct Tolerogenic Phenotype via Contact-Dependent STAT3 Activation

BACKGROUND: Activation of the signal transducer and activator of transcription 3 (STAT3) within antigen presenting cells (APCs) is linked to abnormal APCs differentiation and function. We have previously shown that STAT3 is activated within APC by a novel contact-dependent mechanism, which plays a key role in mediating the immunomodulatory effects of hMSC. In order to better understand the underlying mechanisms that control APC maturation in a contact dependent manner, we extended our observation to tumor cells. Tumors were shown to secrete a variety of tumor-derived factors that activate STAT3 within infiltrating APCs. We now tested whether tumor cells can activate STAT3 within APC using the contact-dependent mechanism, in addition to soluble factors, and compared these two STAT3 activating pathways. PRINCIPAL FINDINGS: We demonstrate that in addition to tumor-derived secreted factors tumor cells activate STAT3 by a mechanism that is based on cell-cell interaction. We further demonstrate that these two STAT3 activating mechanisms differ in their JAK usage and their susceptibility to JSI-124 inhibition thereby representing two distinct pathways. Significantly, although both pathways activate STAT3, they modulate DCs maturation in a different manner that results in disparate phenotypic outcomes. Whereas the soluble-dependent pathway results in an immature phenotype, the contact-dependent pathway results in an apparently mature phenotype. Albeit their mature-like phenotype these latter cells express the tolerogenic markers ILT3 and ILT4 and possess T cell inhibitory activity. SIGNIFICANCE: This data suggests that, in at least certain cellular microenvironments, cell:cell interactions represent a novel way to activate STAT3 signaling, uncouple APC activation events and consequently regulate immunity and tolerance. Significantly, we have now demonstrated that this contact-dependent signaling pathway differs from that mediated by soluble factors and cytokines, inducing disparate phenotypic outcome, suggesting these two mechanisms have different and possibly complementary biological functions

Identification of a Novel Risk Locus for Multiple Sclerosis at 13q31.3 by a Pooled Genome-Wide Scan of 500,000 Single Nucleotide Polymorphisms

Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system with an important genetic component and strongest association driven by the HLA genes. We performed a pooling-based genome-wide association study of 500,000 SNPs in order to find new loci associated with the disease. After applying several criteria, 320 SNPs were selected from the microarrays and individually genotyped in a first and independent Spanish Caucasian replication cohort. The 8 most significant SNPs validated in this cohort were also genotyped in a second US Caucasian replication cohort for confirmation. The most significant association was obtained for SNP rs3129934, which neighbors the HLA-DRB/DQA loci and validates our pooling-based strategy. The second strongest association signal was found for SNP rs1327328, which resides in an unannotated region of chromosome 13 but is in linkage disequilibrium with nearby functional elements that may play important roles in disease susceptibility. This region of chromosome 13 has not been previously identified in MS linkage genome screens and represents a novel risk locus for the disease

Common genetic variants on chromosome 9p21 are associated with myocardial infarction and type 2 diabetes in an Italian population

<p>Abstract</p> <p>Background</p> <p>A genomic region on chromosome 9p21 has been identified as closely associated with increased susceptibility to coronary artery disease (CAD) and to type 2 diabetes (T2D) although the evidence suggests that the genetic variants within chromosome 9p21 that contribute to CAD are different from those that contribute to T2D.</p> <p>We carried out an association case-control study in an Italian population to test the association between two single nucleotide polymorphisms (SNPs) on the 9p21 locus, rs2891168 and rs10811661, previously reported by the PROCARDIS study, and respectively myocardial infarction (MI) and T2D. Our aim was to confirm the previous findings on a larger sample and to verify the independence of their susceptibility effects: rs2891168 associated with MI but not with T2D and rs10811661 associated with T2D but not with MI.</p> <p>Methods</p> <p>Genomic DNA samples of 2407 Italians with T2D (602 patients), who had had a recent MI (600), or had both diseases (600) and healthy controls (605) were genotyped for the two SNPs. The genotypes were determined by allelic discrimination using a fluorescent-based TaqMan assay.</p> <p>Results</p> <p>SNP rs2891168 was associated with MI, but not with T2D and the G-allele odds ratio (OR) was 1.20 (95% CI 1.02-1.41); SNP rs10811661 was associated with T2D, but not with MI, and the T-allele OR was 1.27 (95% CI 1.04-1.55). ORs estimates from the present study and the PROCARDIS study were pooled and confirmed the previous findings, with greater precision.</p> <p>Conclusions</p> <p>Our replication study showed that rs2891168 and rs10811661 are independently associated respectively with MI and T2D in an Italian population. Pooling our results with those reported by the PROCARDIS group, we also obtained a significant result of association with diabetes for rs10811661 in the European population.</p

Erythropoietin Receptor Signaling Is Membrane Raft Dependent

Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3±1.4(SE) vs. 25.6±3.2 aggregates/cell; p≤0.001), accompanied by a >3-fold increase in cluster size (p≤0.001). Raft fraction immunoblotting showed Epo-R translocation to MR after Epo stimulation and was confirmed by fluorescence microscopy in Epo stimulated UT7 cells and primary erythroid bursts. Receptor recruitment into MR was accompanied by incorporation of JAK2, Lyn, and STAT5 and their activated forms. Raft disruption by cholesterol depletion extinguished Epo induced Jak2, STAT5, Akt and MAPK phosphorylation in UT7 cells and erythroid progenitors. Furthermore, inhibition of the Rho GTPases Rac1 or RhoA blocked receptor recruitment into raft fractions, indicating a role for these GTPases in receptor trafficking. These data establish a critical role for MR in recruitment and assembly of Epo-R and signal intermediates into discrete membrane signaling units

Dasatinib impairs long-term expansion of leukemic progenitors in a subset of acute myeloid leukemia cases

A number of signaling pathways might be frequently disrupted in acute myeloid leukemia (AML). We questioned whether the dual SRC/ABL kinase inhibitor dasatinib can affect AML cells and whether differences can be observed with normal CD34+ cells. First, we demonstrated that normal cord blood (CB) CD34+ cells were unaffected by dasatinib at a low concentration (0.5 nM) in the long-term culture on MS5 stromal cells. No changes were observed in proliferation, differentiation, and colony formation. In a subset of AML cases (3/15), a distinct reduction in cell proliferation was observed, ranging from 48% to 91% inhibition at 0.5 nM of dasatinib, in particular, those characterized by BCR–ABL or KIT mutations. Moreover, the inhibitory effects of dasatinib were cytokine specific. Stem cell factor-mediated proliferation was significantly impaired, associated with a reduced phosphorylation of ERK1/2 and STAT5, whereas no effect was observed on interleukin-3 and thrombopoietin-mediated signaling despite SRC activation. In conclusion, this study demonstrates that dasatinib is a potential inhibitor in a subgroup of AML, especially those that express BCR–ABL or KIT mutations

Abrogation of IL-6-mediated JAK signalling by the cyclopentenone prostaglandin 15d-PGJ2 in oral squamous carcinoma cells

Cyclopentenone 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) exerts antineoplastic effects on various types of human cancer. We recently showed that treatment with 15d-PGJ2 induces apoptosis accompanied by downregulation of the oncogenic signal transducer and activator of transcription 3 (Stat3) signalling in human oral squamous cell carcinoma (SCC) cells. The current study examines the effects of 15d-PGJ2 on the epidermal growth factor receptor (EGFR) and Janus Kinase (JAK)-mediated signalling pathways. Inhibition of Stat3 by 15d-PGJ2 was abolished by exogenous stimulation with transforming growth factor alpha (TGF-α), but not interleukin 6 (IL-6), supporting a selective effect of 15d-PGJ2 on IL-6-mediated signalling. Importantly, 15d-PGJ2 selectively abrogated constitutive and IL-6-mediated JAK phosphorylation without affecting EGFR-activated levels. Moreover, the inhibitory effect of 15d-PGJ2 on JAK signalling required the reactive α,β-unsaturated carbon within the cyclopentenone ring. Targeting of JAK signalling using a specific JAK inhibitor also abolished Stat3 phosphorylation and resulted in apoptosis in oral SCC cells. Our findings provide the first evidence for 15d-PGJ2–mediated downregulation of constitutive and IL-6-induced JAK signalling in cancer and support that JAK inhibition and suppression of EGFR-independent Stat3 activation by 15d-PGJ2 represent a promising approach for induction of apoptosis in oral SCC cells