Tissue Transglutaminase-Mediated AT1 Receptor Sensitization Underlies Pro-inflammatory Cytokine LIGHT-Induced Hypertension.

BACKGROUND: Although numerous recent studies have shown a strong link between inflammation and hypertension, the underlying mechanisms by which inflammatory cytokines induce hypertension remain to be fully elucidated. Hypertensive disorders are also associated with elevated pressor sensitivity. Tissue transglutaminase (TG2), a potent cross-linking enzyme, is known to be transcriptionally activated by inflammatory cytokines and stabilize angiotensin II (Ang II) receptor AT1 (AT1R) via ubiquitination-preventing posttranslational modification. Here we sought to investigate the TG2-mediated AT1R stabilization in inflammation-induced hypertension and its functional consequences with a focus on receptor abundance and Ang II responsiveness. METHODS AND RESULTS: Using an experimental model of inflammation-induced hypertension established by introducing the pro-inflammatory tumor necrosis factor cytokine LIGHT, we provide pharmacologic and genetic evidence that TG2 is required for LIGHT-induced hypertension (systolic pressure on day 6: LIGHT = 152.3 ± 7.4 vs. LIGHT+ERW1041E [TG2 inhibitor] = 105.8 ± 13.1 or LIGHT+TG2-/- = 114.3 ± 4.3 mm Hg, P < 0.05, n = 4-5) and renal compromise (urine albumin/creatinine: LIGHT = 0.17 ± 0.05 vs. LIGHT+ERW1041E = 0.03 ± 0.01 or LIGHT+TG2-/- = 0.06 ± 0.01 mg/mg; plasma creatinine: LIGHT = 1.11 ± 0.04 vs. LIGHT+ERW1041E = 0.94 ± 0.04 or LIGHT+TG2-/- = 0.88 ± 0.09 mg/dl; urine volume: LIGHT = 0.23 ± 0.1 vs. LIGHT+ERW1041E = 0.84 ± 0.13 or LIGHT+TG2-/- = 1.02 ± 0.09 ml/24 hour on day 14, P < 0.05, n = 4-5). Our mechanistic studies showed that the TG2-mediated AT1R modification and accumulation (relative renal AT1R level: phosphate-buffered saline [PBS] = 1.23 ± 0.22, LIGHT = 3.49 ± 0.37, and LIGHT+ERW1041E = 1.77 ± 0.46, P < 0.05, n = 3; LIGHT+TG2+/+ = 85.28 ± 36.11 vs. LIGHT+TG2-/- = 7.01 ± 5.68, P < 0.05, n = 3) induced by LIGHT is associated with abrogated ß-arrestin binding (AT1R/associated ß-arrestin ratio: PBS = 2.62 ± 1.07, LIGHT = 38.60 ± 13.91, and LIGHT+ERW1041E = 6.97 ± 2.91, P < 0.05, n = 3; LIGHT+TG2+/+ = 66.43 ± 44.81 vs. LIGHT+TG2-/- = 2.45 ± 1.78, P < 0.01, n = 3) and could be found in renal medulla tubules of kidneys (relative tubular AT1R level: PBS = 5.91 ± 2.93, LIGHT = 92.82 ± 19.54, LIGHT+ERW1041E = 28.49 ± 11.65, and LIGHT+TG2-/- = 0.14 ± 0.10, P < 0.01, n = 5) and the blood vasculature (relative vascular AT1R level: PBS = 0.70 ± 0.30, LIGHT = 13.75 ± 2.49, and LIGHT+ERW1041E = 3.28 ± 0.87, P < 0.01, n = 3), 2 of the tissues highly related to the genesis of hypertension. Our in vitro cellular assays showed that LIGHT stimulation triggered a rapid TG2-dependent increase in the abundance of AT1Rs (relative AT1R level after 2-hour LIGHT treatment: AT1R (WT)+TG2 = 2.21 ± 0.23, AT1R (Q315A)+TG2 = 0.18 ± 0.23, P < 0.05 vs. starting point = 1, n = 2) and downstream calcium signaling (fold increase in NFAT-driven luciferase activity: Saline = 0.02 ± 0.03, Ang II = 0.17 ± 0.08, LIGHT = 0.05 ± 0.04, LIGHT+Ang II = 0.90 ± 0.04 (P < 0.01 vs. Ang II), and LIGHT+Ang II+ERW1041E = 0.15 ± 0.15 (P < 0.01 vs. LIGHT+Ang II), n = 3). CONCLUSIONS: Our data indicate an essential and systemic role for TG2 in bridging inflammation to hypertension via its posttranslational modifications stabilizing AT1 receptor and sensitizing Ang II. Our findings also suggest that TG2 inhibitors could be used as a novel group of cardiovascular agents.

Interleukin-22 reverses human islet dysfunction and apoptosis triggered by hyperglycemia and LIGHT.

Interleukin (IL)-22 has recently been suggested as an anti-inflammatory cytokine that could protect the islet cells from inflammation- and glucose-induced toxicity. We have previously shown that the tumor necrosis factor family member, LIGHT, can impair human islet function at least partly via pro-apoptotic effects. Herein, we aimed to investigate the protective role of IL-22 on human islets exposed to the combination of hyperglycemia and LIGHT. First, we found upregulation of LIGHT receptors (LTßR and HVEM) in engrafted human islets exposed to hyperglycemia (>11 mM) for 17 days post transplantation by using a double islet transplantation mouse model as well as in human islets cultured with high glucose (HG) (20 mM glucose) + LIGHT in vitro, and this latter effect was attenuated by IL-22. The effect of HG + LIGHT impairing glucose-stimulated insulin secretion was reversed by IL-22. The harmful effect of HG + LIGHT on human islet function seemed to involve enhanced endoplasmic reticulum stress evidenced by upregulation of p-IRE1α and BiP, elevated secretion of pro-inflammatory cytokines (IL-6, IL-8, IP-10 and MCP-1) and the pro-coagulant mediator tissue factor (TF) release and apoptosis in human islets, whereas all these effects were at least partly reversed by IL-22. Our findings suggest that IL-22 could counteract the harmful effects of LIGHT/hyperglycemia on human islet cells and potentially support the strong protective effect of IL-22 on impaired islet function and survival.

Trimerization of murine TNF ligand family member LIGHT increases the cytotoxic activity against the FM3A mammary carcinoma cell line.

LIGHT is a member of the tumor necrosis factor ligand superfamily, which plays important roles in inflammatory and immune responses. LIGHT forms a membrane-anchored homotrimeric complex on the cell surface and is often processed as a soluble protein. Recombinant soluble human LIGHT produced by mammalian cells or Escherichia coli is functional at nanomolar concentrations. However, there is little information about the biological activity of mouse LIGHT (mLIGHT) because of the difficulty in producing bioactive soluble mLIGHT. In this study, recombinant trimeric soluble mLIGHT, or Foldon-mLIGHT, was produced by fusing mLIGHT with the trimerization domain foldon from bacteriophage T4 fibritin. Foldon-mLIGHT was secreted from 293F cells as a 68-kDa trimeric protein. The recombinant protein potently inhibited the growth of the FM3A mouse mammary carcinoma cell line with an IC(50) of 77 pM; however, the monomer or dimer forms of mLIGHT produced by E. coli or mammalian cell systems showed weak or no inhibitory activity. These data clearly indicated that trimerization of soluble mLIGHT is essential for its biological activity.