Transglutaminase 2 Inhibitors Research Articles

Extracellular Transglutaminase 2 Is Catalytically Inactive, but Is Transiently Activated upon Tissue Injury

Transglutaminase 2 (TG2) is a multifunctional mammalian protein with transamidase and signaling properties. Using selective TG2 inhibitors and tagged nucleophilic amine substrates, we show that the majority of extracellular TG2 is inactive under normal physiological conditions in cell culture and in vivo. However, abundant TG2 activity was detected around the wound in a standard cultured fibroblast scratch assay. To demonstrate wounding-induced activation of TG2 in vivo, the toll-like receptor 3 ligand, polyinosinic-polycytidylic acid (poly(I:C)), was injected in mice to trigger small intestinal injury. Although no TG2 activity was detected in vehicle-treated mice, acute poly(I:C) injury resulted in rapid TG2 activation in the small intestinal mucosa. Our findings provide a new basis for understanding the role of TG2 in physiology and disease.

ATP-mediated mineralization of MC3T3-E1 osteoblast cultures

While bone is hypomineralized in hypophosphatemia patients and in tissue-nonspecific alkaline phosphatase (Tnsalp)-deficient mice, the extensive mineralization that nevertheless occurs suggests involvement of other phosphatases in providing phosphate ions for mineral deposition. Although the source of phosphate liberated by these phosphatases is unknown, pyrophosphate, ATP, pyridoxal-5′-phosphate (PLP) and phoshoethanolamine (PEA) are likely candidates. In this study, we have induced mineralization of MC3T3-E1 osteoblast cultures using ATP, and have investigated potential phosphatases involved in this mineralization process. MC3T3-E1 osteoblasts were cultured for 12 days and treated either with β-glycerophosphate (βGP) or ATP. Matrix and mineral deposition was examined by biochemical, cytochemical, ultrastructural and X-ray microanalytical methods. ATP added at levels of 4–5 mM resulted in mineral deposition similar to that following conventional treatment with βGP. Collagen levels were similarly normal in ATP-mineralized cultures and transmission electron microscopy and X-ray microanalysis confirmed hydroxyapatite mineral deposition along the collagen fibrils in the ECM. Phosphate release from 4 mM ATP into the medium was rapid and resulted in approximately twice the phosphate levels than after release from 10 mM βGP. ATP treatment did not affect mineralization by altering the expression of mineral-regulating genes such as Enpp1, Ank, and Mgp, nor phosphatase genes indicating that ATP induces mineralization by serving as a phosphate source for mineral deposition. Levamisole, an inhibitor of TNSALP, completely blocked mineralization in βGP-treated cultures, but had minor effects on ATP-mediated mineralization, indicating that other phosphatases such as plasma membrane Ca 2+ transport ATPase 1 (PMCA1) and transglutaminase 2 (TG2) are contributing to ATP hydrolysis. To examine their involvement in ATP-mediated mineralization, the inhibitors cystamine (TG2 inhibitor) and ortho-vanadate (PMCA inhibitor) were added to the cultures — both inhibitors significantly reduced mineralization whereas suppression of the phosphate release by ortho-vanadate was minor comparing to other two inhibitors. The contribution of PMCA1 to mineralization may occur through pumping of calcium towards calcification sites and TG2 can likely act as an ATPase in the ECM. Unlike the GTPase activity of TG2, its ATPase function was resistant to calcium, demonstrating the potential for participation in ATP hydrolysis and mineral deposition within the ECM at elevated calcium concentrations.

Structure−Activity Relationship Analysis of the Selective Inhibition of Transglutaminase 2 by Dihydroisoxazoles

Human transglutaminase 2 (TG2) is believed to play an important role in the pathogenesis of various human disorders including celiac sprue, certain neurological diseases, and some types of cancer. Selective inhibition of TG2 should therefore enable further investigation of its role in physiology and disease and may lead to effective clinical treatment. Recently we showed that certain 3-halo-4-,5-dihydroisoxazole containing compounds are selective inhibitors of human TG2 with promising pharmacological activities. Here, we present definitive evidence that this class of compounds targets the active site of human TG2. Structure-activity relationship studies have provided insights into the structural prerequisites for selectivity and have led to the discovery of an inhibitor with about 50-fold higher activity than a prototypical dihydroisoxazole inhibitor with good in vivo activity. A method for preparing enantiomerically enriched analogues was also developed. Our studies show that the 5-(S)-dihydroisoxazole is a markedly better inhibitor of human TG2 than its 5-(R) stereoisomer.

Transglutaminase differentially regulates growth signalling in rat perivenous and periportal hepatocytes

The influence of transglutaminase 2 (TG2) activity on the proliferative effect of epidermal growth factor (EGF) and on EGF receptor affinity in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) has been investigated using a primary culture system. PPH and PVH subpopulations have been isolated using the digitonin/collagenase perfusion technique. DNA synthesis was assessed by [3H] thymidine incorporation into hepatocytes. The assay for binding of [125I] EGF to cultured hepatocytes was analysed by Scatchard plot analysis. Pretreatment with the TG2 inhibitor monodansylcadaverine (MDC) greatly increased EGF-induced DNA synthesis in both PPH and PVH. Furthermore, [125I] EGF binding studies in PVH treated with MDC indicated that high-affinity EGF receptor expression was markedly up-regulated, whereas in PPH, there was no significant effect. Treatment with retinoic acid (RA), an inducer of TG2 expression, significantly decreased EGF-induced DNA synthesis in both PPH and PVH. Binding studies in the presence of RA revealed that the high-affinity EGF receptor was down-regulated and completely absent in both PPH and PVH. These results suggest that TG2 was involved in the differential growth capacities of PPH and PVH through down-regulation of high-affinity EGF receptors.