Lack of NF-kappaB1 (p105/p50) attenuates unloading-induced downregulation of PPARalpha and PPARalpha-regulated gene expression in rodent heart.

OBJECTIVE: Unloading of the rodent heart activates the fetal gene program, decreases peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARalpha-regulated gene expression (MCAD), and induces cardiomyocyte atrophy. NF-kappaB regulates the fetal gene program and PPARalpha-regulated gene expression during cardiac hypertrophy and induces atrophy in skeletal muscle. Our objective was to test the hypothesis that NF-kappaB is the regulator for activation of the fetal gene program, for downregulation of PPARalpha and PPARalpha-regulated gene expression, and for cardiomyocyte atrophy in the heart subjected to mechanical unloading. METHODS: Activation of the inhibitory kappa B kinase beta (IKKbeta)/NF-kappaB pathways were measured in the heterotopically transplanted rat heart using Western blotting of total and phospho-IKKbeta and using transcription factor ELISA's for the five members of the NF-kappaB family (p65 (Rel A), p105/p50, c-Rel, RelB, and p100/p52). In loss of function experiments, we transplanted hearts of p105/p50 knockout mice into wildtype mice and compared changes in gene expression and cardiomyocyte size with wildtype hearts transplanted into wildtype mice. RESULTS: Total and phospho-IKKbeta levels significantly increased in the transplanted heart seven days after surgery. The activation of IKKbeta was paralleled by increased DNA binding activity of p65 and p105/p50. Mechanical unloading induced myosin heavy chain beta expression and decreased cardiomyocyte size in hearts of both wildtype and p105/p050 knockout animals. In contrast, the downregulation of PPARalpha and MCAD was significantly attenuated or prevented in the hearts of p105/p50 knockout mice. CONCLUSIONS: The IKKbeta/p65/p50 pathway is activated in the unloaded rodent heart and a likely regulator for the downregulation of PPARalpha and PPARalpha-regulated gene expression.

Mechanical unloading of the heart activates the calpain system.

The mechanism for the decrease in cardiomyocyte size with mechanical unloading is unknown. The calpain system regulates cardiomyocyte atrophy. We obtained samples from failing human hearts at the time of implantation and explantation of a left ventricular assist device. For mechanical unloading, we also heterotopically transplanted rat or mouse hearts for 1 week. The effect of calpain inhibition on cardiac atrophy was assessed in transplanted hearts overexpressing calpastatin. We measured transcript levels of calpain 1 and 2 in the human and the rodent model, as well as calpain activity, a calpain-specific degradation product and cardiomyocyte size in the two rodent models. Mechanical unloading of the failing human heart significantly increased calpain 2 gene expression. Transcript levels of calpain 1 and 2, calpain activity and a calpain-specific degradation product all significantly increased in the unloaded rat heart. Unexpectedly, in hearts of animals overexpressing calpastatin, cardiomyocyte size also decreased. Mechanical unloading of the mammalian heart activates the calpain system, although other proteolytic systems may compensate for decreased calpain activity when calpastatin is overexpressed.

The Mannich base NC1153 promotes long-term allograft survival and spares the recipient from multiple toxicities.

JAK3 is a cytoplasmic tyrosine kinase with limited tissue expression but is readily found in activated T cells. Patients lacking JAK3 are immune compromised, suggesting that JAK3 represents a therapeutic target for immunosuppression. Herein, we show that a Mannich base, NC1153, blocked IL-2-induced activation of JAK3 and its downstream substrates STAT5a/b more effectively than activation of the closely related prolactin-induced JAK2 or TNF-alpha-driven NF-kappaB. In addition, NC1153 failed to inhibit several other enzymes, including growth factor receptor tyrosine kinases, Src family members, and serine/threonine protein kinases. Although NC1153 inhibited proliferation of normal human T cells challenged with IL-2, IL-4, or IL-7, it did not block T cells void of JAK3. In vivo, a 14-day oral therapy with NC1153 significantly extended survival of MHC/non-MHC mismatched rat kidney allografts, whereas a 90-day therapy induced transplantation tolerance (>200 days). Although NC1153 acted synergistically with cyclosporin A (CsA) to prolong allograft survival, it was not nephrotoxic, myelotoxic, or lipotoxic and did not increase CsA-induced nephrotoxicity. In contrast to CsA, NC1153 was not metabolized by cytochrome P450 3A4. Thus, NC1153 prolongs allograft survival without several toxic effects associated with current immunosuppressive drugs.

Selection of lowly immunogenic and highly tolerogenic donor and recipient allochimeric class I major histocompatibility complex proteins.

BACKGROUND: Ten different highly polymorphic amino acids (AAs) are located in the alpha1 (alpha1h) and alpha2 (alpha2h) helical regions of the class I major histocompatibility complex RT1. An rat alloantigen. We examined the potential of alpha1h-RT1. An versus alpha2h-RT1. An polymorphic AAs to induce accelerated rejection or tolerance of heart allografts. METHODS: The allochimeric alpha1h52-90n-RT1.Ac and alpha2h148-179n-RT1.Ac cDNAs were produced by the substitution of nucleotides encoding recipient RT1.Ac AAs for donor RT1. An AAs. Allochimeric and wild-type (WT)-RT1. An proteins were generated in an Escherichia coli expression system. RESULTS: A single portal vein administration of 100 mug alpha1h52-90n-RT1.Ac protein in combination with a 7-day course of oral cyclosporine A (4 mg/kg) induced tolerance to Brown Norway (BN) (RT1n) heart allografts in PVG (RT1c) recipients more effectively than did WT-RT1. An protein; alpha2h148-179n-RT1.Ac protein was ineffective. However, subcutaneous injection of 100 mug WT-RT1. An (but neither alpha1h52-90n-RT1.Ac nor alpha2h148-179n-RT1.Ac) protein induced accelerated rejection of BN heart allografts. Untreated PVG recipients of BN heart allografts displayed activation of both interleukin (IL)-2- and interferon-gamma-producing T helper (Th) 1 cells and IL-4- and IL-10-producing Th2 cells on days 5, 7, and 14 postgrafting, as measured by an enzyme-linked immunospot assay. In contrast, in comparison with rejectors, tolerant recipients showed down-regulation of Th1 cells and up-regulation of Th2 cells on days 5, 7, 14, and 200 postgrafting. Histology of heart allografts showed that tolerant BN heart allografts had no evidence of acute or chronic rejection when examined on day 100 after transplantation. CONCLUSIONS: The poorly immunogenic alpha1h52-90n-RT1.Ac allochimeric protein induces tolerance by selective activation of regulatory Th2 cells.

Deoxycorticosterone upregulates PDS (Slc26a4) in mouse kidney: role of pendrin in mineralocorticoid-induced hypertension.

Pendrin is an anion exchanger expressed along the apical plasma membrane and apical cytoplasmic vesicles of type B and of non-A, non-B intercalated cells of the distal convoluted tubule, connecting tubule, and cortical collecting duct. Thus, Pds (Slc26a4) is a candidate gene for the putative apical anion-exchange process of the type B intercalated cell. Because apical anion exchange-mediated transport is upregulated with deoxycorticosterone pivalate (DOCP), we tested whether Pds mRNA and protein expression in mouse kidney were upregulated after administration of this aldosterone analogue by using quantitative real-time polymerase chain reaction as well as light and electron microscopic immunolocalization. In kidneys from DOCP-treated mice, Pds mRNA increased 60%, whereas pendrin protein expression in the apical plasma membrane increased 2-fold in non-A, non-B intercalated cells and increased 6-fold in type B cells. Because pendrin transports HCO3- and Cl-, we tested whether DOCP treatment unmasks abnormalities in acid-base or NaCl balance in Pds (-/-) mice. In the absence of DOCP, arterial pH, systolic blood pressure, and body weight were similar in Pds (+/+) and Pds (-/-) mice. After DOCP treatment, weight gain and hypertension were observed in Pds (+/+) but not in Pds (-/-) mice. Moreover, after DOCP administration, metabolic alkalosis was more severe in Pds (-/-) than Pds (+/+) mice. We conclude that pendrin is upregulated with aldosterone analogues and is critical in the pathogenesis of mineralocorticoid-induced hypertension and metabolic alkalosis.

Allochimeric class I MHC protein-induced tolerance by partial TCR engagement requires activation of both CTL4- and common gamma-chain-dependent cytokine signals.

BACKGROUND: The various toxicities associated with the general immune suppression resulting from current clinical immunosuppressive therapies continue to plague transplant recipients as well as jeopardize allograft survival. METHODS: The present study utilized allochimeric class I MHC antigens (alpha1hu70-77-RT1.Aa) bearing only four donor RT1.Au polymorphic amino acids (a.a.; His70, Val73, Asn74, and Asn77) superimposed on the recipient RT1.Aa background to induce transplantation tolerance in the rat cardiac transplant model. RESULTS: Oral delivery of alpha1hu70-77-RT1.Aa protein alone (days 0-6) induced tolerance, as evidenced by inhibition of both acute and chronic rejection processes. Delivery of alpha1hu70-77-RT1.Aa with therapeutic doses of cyclosporine (CsA) also prevented chronic rejection, otherwise readily developed after treatment with CsA alone. A polymerase chain reaction (PCR)-based analysis showed that tolerant recipients had reduced numbers of interleukin (IL)-2/interferon (IFN)-gamma-producing T helper (Th)1 cells and elevated numbers of IL-4/IL-10-producing Th2 cells. Adoptive transfer experiments revealed that potent regulatory T cells mediated tolerance. The same T cells displayed diminished T cell receptor (TCR)-driven signaling via extracellular regulated kinase, AP-1, and NF-kappaB, as well as the common gamma-chain (gammac) cytokine-receptor-induced signaling by Janus kinase 3 (Jak3)/stimulators and activators of transcription Stat/5 pathways. Tolerance induction was prevented in vivo by inhibition of signal 2 by CTL4Ig or of signal 3 by either rapamycin, which disrupts the mammalian target of rapamycin, or AG490, which inhibits Jak3. Finally, partial or complete tyrosine phosphorylation of Zap70 was observed in alloantigen-specific T cell clones in response to tolerogenic versus immunogenic peptides, respectively. CONCLUSIONS: Tolerance induction by allochimeric proteins is achieved by partial TCR activation in the presence of signals 2 and 3, resulting in a skewed Th2 phenotype.

Selective inhibitor of Janus tyrosine kinase 3, PNU156804, prolongs allograft survival and acts synergistically with cyclosporine but additively with rapamycin.

Janus kinase 3 (Jak3) is a cytoplasmic tyrosine (Tyr) kinase associated with the interleukin-2 (IL-2) receptor common gamma chain (gamma(c)) that is activated by multiple T-cell growth factors (TCGFs) such as IL-2, -4, and -7. Using human T cells, it was found that a recently discovered variant of the undecylprodigiosin family of antibiotics, PNU156804, previously shown to inhibit IL-2-induced cell proliferation, also blocks IL-2-mediated Jak3 auto-tyrosine phosphorylation, activation of Jak3 substrates signal transducers and activators of transcription (Stat) 5a and Stat5b, and extracellular regulated kinase 1 (Erk1) and Erk2 (p44/p42). Although PNU156804 displayed similar efficacy in blocking Jak3-dependent T-cell proliferation by IL-2, -4, -7, or -15, it was more than 2-fold less effective in blocking Jak2-mediated cell growth, its most homologous Jak family member. A 14-day alternate-day oral gavage with 40 to 120 mg/kg PNU156804 extended the survival of heart allografts in a dose-dependent fashion. In vivo, PNU156804 acted synergistically with the signal 1 inhibitor cyclosporine A (CsA) and additively with the signal 3 inhibitor rapamycin to block allograft rejection. It is concluded that inhibition of signal 3 alone by targeting Jak3 in combination with a signal 1 inhibitor provides a unique strategy to achieve potent immunosuppression.