The human phospholamban gene: structure and expression.

Phospholamban, through modulation of sarcoplasmic reticulum calcium-ATPase activity, is a key regulator of cardiac diastolic function. Alterations in phospholamban expression may define parameters of muscle relaxation. In experimental animals, phospholamban is differentially expressed in various striated and smooth muscles, and within the four chambers of the heart. Decreased phospholamban expression within the heart during heart failure has also been observed. Furthermore, regulatory elements of mammalian phospholamban genes remain poorly defined. To extend these studies to humans, we (1) characterized phospholamban expression in various human organs, (2) isolated genomic clones encoding the human phospholamban gene, and (3) prepared human phospholamban promoter/luciferase reporter constructs and performed transient transfection assays to begin identification of regulatory elements. We observed that human ventricle and quadriceps displayed high levels of phospholamban transcripts and proteins, with markedly lower expression observed in smooth muscles, while the right atria also expressed low levels of phospholamban. The human phospholamban gene structure closely resembles that reported for chicken, rabbit, rat, and mouse. Comparison of the human to other mammalian phospholamban genes indicates a marked conservation of sequence for at least 217 bp upstream of the transcription start site, which contains conserved motifs for GATA, CP1/NFY, M-CAT-like, and E-box elements. Transient transfection assays with a series of plasmids containing deleted 5' flanking regions (between -2530 and -66 through +85) showed that sequences between -169 and the CP1-box at -93 were required for maximal promoter activity in neonatal rat cardiomyocytes. Activity of these reporters in HeLa cells was markedly lower than that observed in rat cardiomyocytes, suggesting at least a partial tissue selectivity of these reporter constructs.

Characterization of proximal transcription regulatory elements in the rat phospholamban promoter.

Phospholamban is a major regulator of cardiac diastole, with alterations in expression associated with modified cardiac relaxation. To study transcriptional regulation of phospholamban expression, we made reporter constructs that expressed luciferase under control of putative promoter sequences from the rat phospholamban gene. When transfected into neonatal rat cardiomyocytes, constructs containing at least 159 nucleotides preceding the transcription start site were equally active, while truncation to -66/+64 removed all promoter activity. Constructs were more active in cardiomyocytes than in HeLa cells (which do not express phospholamban), but did not show absolute cell-type specificity of expression. Addition of sequences upstream to -4032, all of the intron (7.4 kb), or 3'UTR sequences (0. 8 kb) did not enhance cell-specific expression. To focus on the basal promoter region (-159/-66), a series of deletion constructs were made that identified a novel 35 bp region (-159/-125; Phospholamban Promoter Element 1, PPE1) required for promoter activity in cardiomyocytes. Site-specific mutations identified nucleotides -150/-133 as containing most of the promoter-enhancing activity. While the rat PPE1 is highly conserved (>70%) in four other mammalian phospholamban genes, it does not contain previously characterized regulatory elements. In cardiomyocytes the PPE1 sequence markedly enhanced activity of the SV40 early promoter. A conserved CCAAT element (-83/-79) was also required for promoter activity in both cardiomyocytes and HeLa cells. Exonuclease III footprinting identified protein/DNA interactions in both the extended CCAAT box and PPE1 domains. Gel shift studies identified the CCAAT elements as binding CBF/NF-Y.

Interleukin-1 beta inhibits phospholamban gene expression in cultured cardiomyocytes.

Phospholamban is a key regulatory protein that defines diastolic function. Proinflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) can depress contractility and intracellular Ca2+ currents and transients. An alteration in phospholamban expression is a possible pathway by which these cytokines modulate cardiac function. To test this hypothesis, primary cultures of neonatal rat cardiomyocytes were incubated with IL-1 beta, TNF-alpha, or both, and the level of phospholamban transcripts was examined by Northern blot analyses. Phospholamban transcript levels were decreased approximately equal to 50% (P < .0001) in cells exposed to 2 ng/mL IL-1 beta (20 hours), whereas TNF-alpha had no effect. Western blot analyses showed that IL-1 beta also reduced phospholamban protein levels (60% of control, P < .0001). The effects on transcript levels were gene specific; IL-1 beta induced transcripts for inducible NO synthase (iNOS), did not alter GAPDH transcripts, and reduced sarcoplasmic reticulum Ca(2+)-ATPase (65% of control, P < .001) transcripts. Cardiomyocytes treated with IL-1 beta showed no alterations in basal contractile parameters (maximum velocity of contraction and relaxation and maximal amplitude of contraction) but were unresponsive to beta-adrenergic stimulation. Studies performed in the presence of second-messenger inhibitors showed that the effect of IL-1 beta on phospholamban transcript levels was blocked by dexamethasone, was insensitive to inhibitors of iNOS, cyclooxygenase, or tyrosine kinases, but was enhanced by the addition of the protein kinase inhibitor staurosporine. These data demonstrate that IL-1 beta alters the expression of phospholamban, a key regulator of cardiac contractility, at both the transcript and protein levels. The results suggest novel mechanisms by which IL-1 beta may modify cardiac function.

Dilated cardiomyopathy in transgenic mice with cardiac-specific overexpression of tumor necrosis factor-alpha.

The failing human heart expresses tumor necrosis factor-alpha (TNF-alpha). However, its pathophysiological significance is not clear. We previously reported that robust overexpression of TNF-alpha in the murine heart causes lethal myocarditis. In this study, we modified the transgene to reduce the production of TNF-alpha by preserving the destabilizing sequence in TNF-alpha cDNA. Expression was driven by the murine alpha-myosin heavy chain promoter. Use of this modified construct allowed to the establish a mutine transgenic line (TG). TG offspring were examined at 6, 12, and 24 weeks. All showed a significantly higher heart weight-to-body weight ratio. Northern blot analysis confirmed the expression of transgene in the heart, and enzyme-linked immunosorbent assay demonstrated the presence of TNF-alpha protein. The TG heart demonstrated a mild, diffuse, lymphohistiocytic interstitial inflammatory infiltrate. Cardiomyocyte necrosis and apoptosis were present but not abundant. Magnetic resonance imaging showed that the TG heart was significantly dilated with reduced ejection fraction. Although the left ventricular dP/dtmax was not different at baseline, its responsiveness to isoproterenol was significantly blunted in TG. Atrial natriuretic factor was expressed in the TG ventricle. A group of TG died spontaneously, and subsequent autopsies revealed exceptional dilation of the heart, increased lung weight, and pleural effusion, suggesting that they died of congestive heart failure. The cumulative mortality rate at 6 months was 23%. In conclusion, the mouse overexpressing TNF-alpha recapitulated the phenotype of congestive heart failure. This provides a novel model to elucidate the role of this cytokine in the development of congestive heart failure.

IL-8/IL-8 receptor expression in psoriasis and the response to systemic tacrolimus (FK506) therapy.

Recently, the keratinocyte IL-8/IL-8 receptor (IL-8R) pathway has been implicated in the pathogenesis of psoriasis, and there is evidence that the potent macrolide immune suppressant tacrolimus (formerly FK506) can inhibit this pathway in vitro. In this study, determination of the expression of cytokine mRNAs in lesional skin of patients with active disease by reverse transcriptase polymerase chain reaction revealed transcripts for IL-1 beta, tumour necrosis factor-alpha (TNF-alpha), IL-6, IL-8, IL-8R, IL-10, interferon-gamma (IFN-gamma), IL-2R and transforming growth factor-beta (TGF-beta), but not IL-2 or IL-4. IL-8 was the only cytokine expressed in affected skin of all patients but not in clinically normal skin of healthy subjects. In seven CD4+ T cell clones propagated from the lesional skin of an untreated psoriasis patient, IL-8 was expressed by the skin-derived T lymphocytes and not by feeder cells (irradiated autologous blood lymphocytes); IL-1 beta, IL-2, IL-6 and IL-10 were also expressed by some or all of the T cell clones. IL-8 mRNA was not detected in the skin of any patient after the start of systemic tacrolimus therapy; IL-1 beta, IL-6 and IFN-gamma transcripts were also reduced. By 12 weeks, the mean psoriasis area and severity index (PASI) had decreased from 18.8 to 3.8, a reduction of 80%. In the same post-treatment biopsies, however, message for IL-8R persisted. Estimation of circulating IL-8 levels by enzyme immunoassay showed that all patients with detectable IL-8 before treatment had decreased levels in response to treatment with tacrolimus; reductions in PASI scores were accompanied by decreases in IL-8 levels, that varied both in rate and extent. Partial relapse, which in a minority of patients followed the initial period of remission, and was precipitated by drug dose reduction, was accompanied by an increase in circulating IL-8. These findings add credence to the view that the IL-8/IL-8R autocrine/paracrine pathway may be important in the pathogenesis of psoriasis. They further suggest that interference with IL-8 production and/or that of other key chemokines may be an important mechanism underlying the therapeutic efficacy of tacrolimus, and other agents such as cyclosporin A, with similar molecular actions.