Kip, Sertac N.

In airway smooth muscle (ASM), the state of sarcoplasmic reticulum (SR) Ca 2+ stores is an important determinant of [Ca 2+ ] i response to agonist. We have previously shown that acetylcholine (ACh) induces sustained [Ca 2+ ] i oscillations representing repetitive SR Ca 2+ release and reuptake. Thus, SR refilling is a key component in refilling of SR stores. We hypothesized that, like cardiac and vascular smooth muscle, ASM SR refilling is regulated by calmodulin (CaM)‐CaM kinase (CaMKII) dependent phosphorylation of phospholamban (PLB). In ASM cells from fresh porcine trachea, effects of W7 (CaM antagonist) and KN‐93/KN‐62 (CaMKII antagonists) on [Ca 2+ ] i responses to ACh were measured using fluorescence microscopy. Separately, SR‐enriched membranes were isolated to monitor ATP‐energized Ca 2+ uptake and in vitro phosphorylation of PLB in the presence of CaMKII inhibitors. KN‐93 significantly slowed decay of [Ca 2+ ] i responses, and slowed or inhibited ongoing [Ca 2+ ] i oscillations, with visible slowing of fall‐times of individual oscillations. Pre‐exposure to W7 or KN‐62 did not prevent initiation of oscillations. In SR‐enriched membranes, addition of KN‐93/KN‐62 resulted in significantly slower Ca 2+ uptake, accompanied by a matching decrement in the in vitro phosphorylation of Thr 17 ‐PLB, indicating the influence of CaM‐CaMKII on this protein. These data suggest that agonist‐induced [Ca 2+ ] i regulation in ASM involves the CaM‐CaMKII pathway which indirectly influences Ca 2+ release and reuptake, likely through PLB. Supported by NIH grant HL74309 (GCS)

Abstract Abstract 88 Introduction: The biology and genetics of anaplastic large T-cell lymphomas (ALCLs) lacking ALK translocations remain poorly defined. We recently identified recurrent translocations involving the DUSP22-IRF4 locus on 6p25.3 in ALK-negative ALCL (Blood 2011;117:915–9). This translocation is present in about 20% of ALK-negative ALCLs and is absent in other T-cell lymphomas; however, the biologic consequences are unknown. In this study, we utilized gene expression profiling (GEP) to compare T-cell lymphomas with and without this translocation. Methods: cDNA was prepared from total RNA extracted from frozen tissue from 25 T-cell lymphoma patients under a Mayo Clinic IRB-approved protocol as follows: 5 ALK-negative ALCLs with 6p25.3 translocations (2 systemic, 3 cutaneous), 12 ALK-negative ALCLs without 6p25.3 translocations (11 systemic, 3 cutaneous), 5 ALK-positive ALCLs, and 3 CD30-positive peripheral T-cell lymphomas (1 transformed mycosis fungoides and 2 not otherwise specified). Partner loci in cases with 6p25.3 translocations were 7p32.3 in 2, 9p24.3 in 1, and unknown in 2. GEP data (Affymetrix U133 plus 2.0) were analyzed by unsupervised clustering; in addition, a GEP signature was developed using genes with raw values >200 in at least 2 cases with at least 2-fold, statistically significantly different expression between 6p25.3-translocated and non-translocated cases (p<0.05 after Benjamini-Hochberg multiple testing correction). Representative overexpressed genes were confirmed by quantitative real-time (qRT-) PCR. Supervised pathway analysis was performed using signatures derived from other studies or curated from public pathway databases. Results: By unsupervised clustering, the 5 cases with 6p25.3 translocations clustered together (281 probe sets). A GEP signature segregating translocated from non-translocated cases included 283 genes (195 up- and 88 down-regulated). Key features of this signature included altered expression of histone-modifying genes (e.g. EZH2, SUZ12, MLLT3, MLL; HDAC4) and up-regulation of cancer-testis antigen (CTA) genes (e.g. SSX4, TEX14, MAGEA12, SSX1, CTAG2). The signature included down-regulation of DUSP22 but not of other genes on 6p25 in the signature. Additional overexpressed genes were validated by qRT-PCR (relative expression: TEX14, 2.9, p=0.01, t test; MAL, 6.5, p=0.003; CCR8, 7.6, p=0.0004). Supervised pathway analysis demonstrated a high proliferation signature in cases with 6p25.3 translocations (p=0.006 vs. untranslocated cases). Conclusions: ALK-negative ALCLs with translocations involving the DUSP22-IRF4 locus on 6p25.3 have a distinct gene expression signature characterized by alterations of histone-modifying genes and CTA genes (which typically are regulated epigenetically, either through histone modifications or changes in methylation). This signature appears independent of anatomic site (systemic vs. cutaneous) and partner locus (7p32.3 vs. other). DUSP22, encoding a dual-specificity phosphatase involved in modulating mitogen-activated protein kinase (MAPK) signaling, is significantly down-regulated in translocated cases; however, pathway analysis did not show significant alterations in MAPK-associated genes. Other known genes in the region of the breakpoint on 6p25 were not affected substantially. These findings suggest the possibility of an unrecognized function of DUSP22. Contributions of the varying partner loci remain unclear. The distinct GEP signature associated with 6p25.3 translocations provides strong support for their biologic relevance. Finally, the genes involved in the histone-modifying signature show overlap with histone-modifying genes mutated in B-cell lymphomas, suggesting potential commonalities in the biology and possibly targeted treatment of B- and T-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

Plasma membrane Ca2+ATPases (PMCAs) are ubiquitous in Ca2+-transporting organs, including the kidney. Using RT-PCR, we detected PMCA1b, PMCA2b (rare), and PMCA4b in Madin-Darby canine kidney (MDCK) cells. At the protein level, only PMCA1 and PMCA4 were readily detected and were highly enriched in the basolateral membrane. The Na+/Ca2+exchanger NCX1 was also detected at the transcript and protein level. A functional assay measuring45Ca2+flux across MDCK cell monolayers under resting conditions indicated that two-thirds of apicobasolateral Ca2+transport was provided by Na+/Ca2+exchanger and one-third by PMCAs, as determined in Na+-free media and using various PMCA inhibitors (La3+, vanadate, calmidazolium, and trifluoroperazine). The importance of PMCA4b for basolateral Ca2+efflux was demonstrated by overexpression of PMCA4b or antisense knockdown of endogenous PMCA4b. Overexpression of PMCA4b increased apicobasolateral Ca2+transport to ∼140%, whereas antisense treatment reduced Ca2+flux ∼45% compared with controls. The MDCK system is thus an ideal model for functional studies of the specific role and regulation of PMCA isoforms in Ca2+reabsorption in the distal kidney.

Cardiovascular complications are the leading cause of morbidity and mortality in autosomal dominant polycystic kidney disease. Pkd2 + /− vascular smooth muscle cells (VSMCs) have an abnormal phenotype and defective intracellular Ca 2+ ([Ca 2+ ] i ) regulation. We examined cAMP content in vascular smooth muscles from Pkd2 + /− mice because cAMP is elevated in cystic renal epithelial cells. We found cAMP concentration was significantly increased in Pkd2 + /− vessels compared with wild-type vessels. Furthermore, reducing the wild-type VSMC [Ca 2+ ] i by Verapamil or BAPTA-AM significantly increased cellular cAMP concentration (mainly by phosphodiesterase [PDE] inhibition), the rate of VSMC proliferation (determined by direct cell counting, 3 H-incorporation, FACS analysis of cells entering S phase, and quantitative Western PCNA and ERK1/2 analyses), and the rate of apoptosis (by Hoechst staining, FACS analysis of the Annexin-V positive cells, and quantitative Western Bax, cytochrome c , and activated caspase 9 and 3 analyses). The low [Ca 2+ ] i induced VSMC proliferation was independent of cAMP/B-Raf signaling, while that of apoptosis was promoted by cAMP. In summary, Pkd2 + /− VSMCs have elevated cAMP levels. This elevation can also be induced by reducing [Ca 2+ ] i in wild-type VSMCs. The [Ca 2+ ] i reduction and cAMP accumulation can cause an increase in both cellular proliferation and apoptosis, resembling Pkd mutant phenotype.

Plasma membrane Ca2+-ATPases (PMCAs) are a ubiquitous system for the expulsion of Ca2+from eukaryotic cells. In tight monolayers of polarized Madin-Darby canine kidney (MDCK) cells representing a distal kidney tubule model, PMCAs are responsible for about one-third of the vectorial Ca2+transport under resting conditions, with the remainder being provided by the Na+/Ca2+exchanger. Vitamin D3(VitD) is known to increase PMCA expression and activity in Ca2+-transporting tissues such as the intestine, as well as in osteoblasts and Madin-Darby bovine kidney epithelial cells. We found that VitD upregulated the expression of the PMCAs (mainly PMCA4b) in MDCK cell lysates at the RNA and protein level in a time- and dose-dependent manner. Interestingly, VitD caused a decrease of the PMCAs in the apical plasma membrane fraction and a concomitant increase of the pumps in the basolateral membrane. Functional studies demonstrated that transcellular45Ca2+flux from the apical-to-basolateral compartment was significantly enhanced by VitD. These findings demonstrate that VitD is a positive regulator of the PMCAs in MDCK epithelial cells. The correlation of decreased apical/increased basolateral expression of the PMCAs with an increase in transcellular Ca2+flux from the apical (urine) toward the basolateral (blood) compartment indicates the physiological relevance of VitD function in kidney tubular Ca2+reabsorption.