Simultaneous attenuation of hyperglycemic memory-induced retinal, pulmonary, and glomerular dysfunctions by proinsulin C-peptide in diabetes.

BACKGROUND: Hyperglycemic memory (HGM) is a pivotal phenomenon in the development of diabetic complications. Although coincident diabetic complications are reported, research on their development and treatment is limited. Thus, we investigated whether C-peptide can simultaneously inhibit HGM-induced retinal, pulmonary, and glomerular dysfunctions in diabetic mice supplemented with insulin. METHODS: Insulin-treated diabetic mice were supplemented with human C-peptide by subcutaneous implantation of K9-C-peptide depots for 4 weeks, and reactive oxygen species (ROS) generation, transglutaminase (TGase) activity, and vascular leakage were examined in the retina, lung, and kidney. RESULTS: We found hyperglycemia-induced persistent ROS generation and TGase activation after blood glucose normalization in the retina, lung, and kidney of insulin-supplemented diabetic mice. These pathological events were inhibited by systemic supplementation of human C-peptide via subcutaneous implantation of a thermosensitive biopolymer-conjugated C-peptide depot. ROS generation and TGase activation were in a vicious cycle after glucose normalization, and C-peptide suppressed the vicious cycle and subsequent endothelial permeability in human retinal endothelial cells. Moreover, C-peptide supplementation ameliorated HGM-induced retinal vascular leakage and neurodegeneration, pulmonary vascular leakage and fibrosis, and glomerular adherens junction disruption and vascular leakage. CONCLUSIONS: Overall, our findings demonstrate that C-peptide supplementation simultaneously attenuates vascular and neuronal dysfunctions in the retina, lung, and glomerulus of insulin-supplemented diabetic mice.

Transglutaminase 2 Inhibitor LDN 27219 Age-Dependently Lowers Blood Pressure and Improves Endothelium-Dependent Vasodilation in Resistance Arteries.

Transglutaminase 2 (TG2) is an enzyme which in the open conformation exerts transamidase activity, leading to protein cross-linking and fibrosis. In the closed conformation, TG2 participates in transmembrane signaling as a G protein. The unspecific transglutaminase inhibitor cystamine causes vasorelaxation in rat resistance arteries. However, the role of TG2 conformation in vascular function is unknown. We investigated the vascular effects of selective TG2 inhibitors by myography in isolated rat mesenteric and human subcutaneous resistance arteries, patch-clamp studies on vascular smooth muscle cells, and blood pressure measurements in rats and mice. LDN 27219 promoted the closed TG2 conformation and inhibited transamidase activity in mesenteric arteries. In contrast to TG2 inhibitors promoting the open conformation (Z-DON, VA5), LDN 27219 concentration-dependently relaxed rat and resistance human arteries by a mechanism dependent on nitric oxide, large-conductance calcium-activated and voltage-gated potassium channels 7, lowering blood pressure. LDN 27219 also potentiated acetylcholine-induced relaxation by opening potassium channels in the smooth muscle; these effects were abolished by membrane-permeable TG2 inhibitors promoting the open conformation. In isolated arteries from 35- to 40-week-old rats, transamidase activity was increased, and LDN 27219 improved acetylcholine-induced relaxation more than in younger rats. Infusion of LDN 27219 decreased blood pressure more effectively in 35- to 40-week than 12- to 14-week-old anesthetized rats. In summary, pharmacological modulation of TG2 to the closed conformation age-dependently lowers blood pressure and, by opening potassium channels, potentiates endothelium-dependent vasorelaxation. Our findings suggest that promoting the closed conformation of TG2 is a potential strategy to treat age-related vascular dysfunction and lowers blood pressure.

Transglutaminase Type 2 is Involved in the Hematopoietic Stem Cells Homeostasis.

Type 2 transglutaminase (TG2) is a multifunctional protein involved in various biological processes playing a key regulatory role in cell homeostasis such as cell death and autophagy. New evidence is emerging that support an important role of autophagy in regulating normal hematopoiesis. Prompted by these findings, in this study we investigated in vivo involvement of TG2 in mouse hematopoiesis under normal or nutrient deprivation conditions. We found that the number and rate of differentiation of bone marrow hematopoietic stem cell was decreased in the TG2 knockout mice. We present evidence showing that these effects on hematopoietic system are very likely due to the TG2-dependent impairment of autophagy. In fact, stimulation of autophagy by starvation is able to rescue the block of the differentiation of stem cells progenitors in the TG2 KO mice. It was also shown that the RhoA/ERK½ pathway, known to be essential for regulation of the bone marrow progenitor cells homeostasis, was significantly impaired in the absence of TG2. Hence, this study expanded our knowledge about TG2 discovering a role of this enzyme in regulation of hematopoiesis.

Transglutaminases and Obesity in Humans: Association of F13A1 to Adipocyte Hypertrophy and Adipose Tissue Immune Response.

Transglutaminases TG2 and FXIII-A have recently been linked to adipose tissue biology and obesity, however, human studies for TG family members in adipocytes have not been conducted. In this study, we investigated the association of TGM family members to acquired weight gain in a rare set of monozygotic (MZ) twins discordant for body weight, i.e., heavy-lean twin pairs. We report that F13A1 is the only TGM family member showing significantly altered, higher expression in adipose tissue of the heavier twin. Our previous work linked adipocyte F13A1 to increased weight, body fat mass, adipocyte size, and pro-inflammatory pathways. Here, we explored further the link of F13A1 to adipocyte size in the MZ twins via a previously conducted TWA study that was further mined for genes that specifically associate to hypertrophic adipocytes. We report that differential expression of F13A1 (ΔHeavy-Lean) associated with 47 genes which were linked via gene enrichment analysis to immune response, leucocyte and neutrophil activation, as well as cytokine response and signaling. Our work brings further support to the role of F13A1 in the human adipose tissue pathology, suggesting a role in the cascade that links hypertrophic adipocytes with inflammation.

Physiological mechanism of transglutaminase-mediated improvement in salt tolerance of cucumber seedlings.

Transglutaminase (TGase) is inextricably associated with plant growth and development. However, the mechanism by which TGase enhances salt tolerance of higher plants under salt stress is poorly understood. In this study, we investigated the effects of NaCl stress and exogenous o-phenanthroline (o-Phen, a metalloprotease inhibitor) on TGase activity, chlorophyll fluorescence parameters, carbohydrates contents, the reactive oxygen species (ROS) scavenging system, and endogenous polyamines (PAs) contents of salt-sensitive 'Jinyou No. 4' and salt-tolerant 'Inbred Line 9930' cucumber. Salt stress significantly inhibited plant growth of the two cultivars, as well as hindered carbohydrates transport, which was more evident in the salt-sensitive cultivar. TGase activity and expression, ROS scavenging capacity, and bound PAs content were up-regulated by salt stress to some extent, which was more distinct in the salt-tolerant cucumber cultivar. However, o-Phen treatment significantly inhibited TGase expression, and further decreased plant growth and the actual photochemical efficiency of photosystem II in the two cultivars. In addition, application of o-Phen significantly decreased endogenous PAs content in leaves of 'Jinyou No. 4' and 'Inbred Line 9930' seedlings by 9.60% and 42.32% under NaCl stress, respectively. These results suggested that high activity of TGase increases the salt stress tolerance of cucumber plants by increasing endogenous PAs content and ROS scavenging capacity, and promoting carbon assimilation and photosynthetic products.

The expression levels of CHI3L1 and IL15Rα correlate with TGM2 in duodenum biopsies of patients with celiac disease.

OBJECTIVE AND DESIGN: Celiac disease (CD) is an intestinal inflammatory disorder of the small intestine. Gliadins are a component of gluten and there are three main types (α, γ, and ω). Recent studies indicate that gliadin peptides are able to activate an innate immune response. IL15 is a major mediator of the innate immune response and is involved in the early alteration of CD mucosa. The chitinase molecules are highly expressed by the innate immune cells during the inflammatory processes. MATERIAL OR SUBJECTS: We analyzed several microarray datasets of PBMCs and duodenum biopsies of CD patients and healthy control subjects (HCs). We verified the modulation CHI3L1 in CD patients and correlated the expression levels to the IL15, IL15Rα, TGM2, IFNγ, and IFNGR1/2. Duodenal biopsy samples belonged to nine active and nine treated children patients (long-term effects of gliadin), and 17 adult CD patients and 10 adults HCs. We also selected 169 samples of PBMCs from 127 CD patients on adherence to a gluten-free diet (GFD) for at least 2 years and 44 HCs. RESULTS: Our analysis showed that CHI3L1 and IL15Rα were significantly upregulated in adult and children's celiac duodenum biopsies. In addition, the two genes were correlated significantly both in children than in adults CD duodenum biopsies. No significant modulation was observed in PBMCs of adult CD patients compared to the HCs. The correlation analysis of the expression levels of CHI3L1 and IL15Rα compared to TGM showed significant values both in adults and in children duodenal biopsies. Furthermore, the IFNγ expression levels were positively correlated with CHI3L1 and IL15Rα. Receiver operating characteristic (ROC) analysis confirmed the diagnostic ability of CHI3L1 and IL15Rα to discriminate CD from HCs. CONCLUSION: Our data suggest a role for CHI3L1 underlying the pathophysiology of CD and represent a starting point aiming to inspire new investigation that proves the possible use of CHI3L1 as a diagnostic factor and therapeutic target.

Ectopic expression of CsTGase enhances salt tolerance by regulating polyamine biosynthesis, antioxidant activities and Na+/K+ homeostasis in transgenic tobacco.

Transglutaminases (TGases), mediators of the transamidation of specific proteins by polyamines (PA), play critical roles in PA metabolism in animals, but their functions and regulatory mechanisms are largely unknown in plants. In this study, we demonstrated that TGase from cucumber played a protective role in the regulation of PA metabolism under salt stress. The expression of TGase was induced by salt stress in cucumber. Ectopic overexpression of cucumber TGase in tobacco conferred enhanced tolerance to salt stress based on both external symptoms and membrane integrity. Overexpression lines maintained high levels of PAs under salt stress, suggesting that PAs played a vital role in TGase-induced salt tolerance. In contrast, the levels of Na+ content in the wild-type (WT) plants increased, while they decreased in the overexpression plants. The expression levels of several genes related to ion exchange enhanced, and the Na+/K+ ratio decreased by increased TGase activity under salt stress. The activities of the proton-pump ATPase (H+-ATPase), vacuolar H+-ATPase (V-ATPase) and vacuolar H+-pyrophosphatase (PPase) were higher in the overexpression lines than in WT plants under salt stress. Moreover, the malondialdehyde (MDA) and H2O2 contents were significantly lower in the overexpression lines than in WT plants, accompanied by increased antioxidant enzyme activity. Taken together, these findings demonstrate that TGase plays protective roles in response to salt stress, which may promote plant survival by regulating PA metabolism and the Na+/K+ balance under salt stress.

Analysis of the expression of transglutaminases in the reconstructed human epidermis using a three-dimensional cell culture.

The skin epidermis functions as a barrier to various external stresses. In the outermost layer, the terminally differentiated keratinocytes result in cornification with a tough structure by formation of a cornified envelope beneath the plasma membrane. To complete the formation of the cornified envelope, several structural proteins are cross-linked via the catalytic action of transglutaminases (TG1, TG3, TG5, and TG6). The expression and activation of these enzymes are regulated in a tightly coordinated manner during keratinocyte differentiation. We here show the system detecting the activity of the TGases using specific glutamine-donor substrate peptides in a three-dimensional culture system of keratinocytes. In this review, we summarize the roles of the epidermal enzymes and introduce a detection method that will provide a system for evaluating the skin barrier function.

Insulin prevents pulmonary vascular leakage by inhibiting transglutaminase 2 in diabetic mice.

AIMS: Insulin is a central peptide hormone required for carbohydrate metabolism; however, its role in diabetes-associated pulmonary disease is unknown. Here, we investigated the preventative effect of insulin against hyperglycemia-induced pulmonary vascular leakage and its molecular mechanism of action in the lungs of diabetic mice. MAIN METHODS: Vascular endothelial growth factor (VEGF) activated transglutaminase 2 (TGase2) by sequentially elevating intracellular Ca2+ and reactive oxygen species (ROS) levels in primary human pulmonary microvascular endothelial cells (HPMVECs). KEY FINDINGS: Insulin inhibited VEGF-induced TGase2 activation, but did not affect intracellular Ca2+ elevation and ROS generation. Insulin prevented VEGF-induced vascular leakage by inhibiting TGase2-mediated c-Src phosphorylation, disassembly of VE-cadherin and ß-catenin, and stress fiber formation. Insulin replacement therapy prevented hyperglycemia-induced TGase2 activation, but not ROS generation, in the lungs of diabetic mice. Insulin also prevented vascular leakage and cancer metastasis in the diabetic lung. Notably, vascular leakage was not detectable in the lungs of TGase2-null (Tgm2-/-) diabetic mice. SIGNIFICANCE: These findings demonstrate that insulin prevents hyperglycemia-induced pulmonary vascular leakage in diabetic mice by inhibiting VEGF-induced TGase2 activation rather than ROS generation.

Magnetic Resonance Imaging Reveals Distinct Roles for Tissue Transglutaminase and Factor XIII in Maternal Angiogenesis During Early Mouse Pregnancy.

OBJECTIVE: The early embryo implantation is characterized by enhanced uterine vascular permeability at the site of blastocyst attachment, followed by extracellular-matrix remodeling and angiogenesis. Two TG (transglutaminase) isoenzymes, TG2 (tissue TG) and FXIII (factor XIII), catalyze covalent cross-linking of the extracellular-matrix. However, their specific role during embryo implantation is not fully understood. Approach and Results: For mapping the distribution as well as the enzymatic activities of TG2 and FXIII towards blood-borne and resident extracellular-matrix substrates, we synthetized selective and specific low molecular weight substrate analogs for each of the isoenzymes. The implantation sites were challenged by genetically modifying the trophoblast cells in the outer layer of blastocysts, to either overexpress or deplete TG2 or FXIII, and the angiogenic response was studied by dynamic contrast-enhanced-magnetic resonance imaging. Dynamic contrast-enhanced-magnetic resonance imaging revealed a decrease in the permeability of decidual vasculature surrounding embryos in which FXIII were overexpressed in trophoblast cell. Reduction in decidual blood volume fraction was demonstrated when either FXIII or TG2 were overexpressed in embryonic trophoblast cell and was elevated when trophoblast cell was depleted of FXIII. These results were corroborated by histological analysis. CONCLUSIONS: In this study, we report on the isoenzyme-specific roles of TG2 and FXIII during the early days of mouse pregnancy and further reveal their involvement in decidual angiogenesis. Our results reveal an important magnetic resonance imaging-detectable function of embryo-derived TG2 and FXIII on regulating maternal angiogenesis during embryo implantation in mice.Visual Overview: An online visual overview is available for this article.

Proinsulin C-peptide prevents hyperglycemia-induced vascular leakage and metastasis of melanoma cells in the lungs of diabetic mice.

C-peptide has a beneficial effect against diabetic complications, but its role in hyperglycemia-induced metastasis is unknown. We investigated hyperglycemia-mediated pulmonary vascular leakage and metastasis and C-peptide inhibition of these molecular events using human pulmonary microvascular endothelial cells (HPMVECs) and streptozotocin-induced diabetic mice. VEGF, which is elevated in the lungs of diabetic mice, activated transglutaminase 2 (TGase2) in HPMVECs by sequential elevation of intracellular Ca2+ and reactive oxygen species (ROS) levels. VEGF also induced vascular endothelial (VE)-cadherin disruption and increased the permeability of endothelial cells, both of which were prevented by the TGase inhibitors monodansylcadaverine and cystamine or TGM2-specific small interfering RNA. C-peptide prevented VEGF-induced VE-cadherin disruption and endothelial cell permeability through inhibiting ROS-mediated activation of TGase2. C-peptide supplementation inhibited hyperglycemia-induced ROS generation and TGase2 activation and prevented vascular leakage and metastasis in the lungs of diabetic mice. The role of TGase2 in hyperglycemia-induced pulmonary vascular leakage and metastasis was further demonstrated in diabetic Tgm2-/- mice. These findings demonstrate that hyperglycemia induces metastasis, and C-peptide prevents the hyperglycemia-induced metastasis in the lungs of diabetic mice by inhibiting VEGF-induced TGase2 activation and subsequent vascular leakage.-Jeon, H.-Y., Lee, Y.-J., Kim, Y.-S., Kim, S.-Y., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. Proinsulin C-peptide prevents hyperglycemia-induced vascular leakage and metastasis of melanoma cells in the lungs of diabetic mice.

Transglutaminase-mediated cross-linking of WDR54 regulates EGF receptor-signaling.

WDR54 is a member of the WD40 repeat (WDR) domain-containing protein family that was recently identified as a novel oncogene in colorectal cancer. However, the molecular mechanism of WDR54 and its functional association with other molecules related to tumor cell growth are unknown. Here, we show that WDR54 can be cross-linked by the action of transglutaminase (TG) 2, which enhances the activation of EGF receptor-mediated signaling pathway. The most carboxyl-terminal WD domain was required for cross-linking. In addition, lysine 280 in WDR54, also in this WD domain, was an important residue for both cross-linking and ubiquitination. Cross-linked WDR54 was found in vesicles aggregated at the plasma membrane. The activated EGF receptor was co-localized with this vesicle, and the internalization of the EGF receptor into the cytosol was sustained. As a result, Erk activity in response to EGF stimulation was enhanced. Furthermore, the growth of the cells lacking WDR54 expression generated by genome editing was delayed compared with that in wild-type cells. Because TG2 is also has been proposed to activate the EGF receptor-signaling and proliferation of tumor cells, WDR54 might have a functional relationship with the EGF receptor and TG2. Our study on the mechanism of biological function of WDR54 may provide rationale for the design and development of a cancer drug based on inhibiting the post-translational modification of this oncogene product.

Knockdown of transglutaminase-2 prevents early age-induced vascular changes in mice1.

PURPOSE: To determine whether the absence of transglutaminase 2 enzyme (TG2) in TG2 knockout mice (TG2-/-) protect them against early age-related functional and histological arterial changes. METHODS: Pulse wave velocity (PWV) was measured using non-invasive Doppler and mean arterial pressure (MAP) was measured in awake mice using tail-cuff system. Thoracic aortas were excised for evaluation of endothelial dependent vasodilation (EDV) by wire myography, as well as histological analyses. RESULTS: PWV and MAP were similar in TG2-/-mice to age-matched wild type (WT) control mice. Old WT mice exhibited a markedly attenuated EDV as compared to young WT animals. The TG2-/-young and old mice had enhanced EDV responses (p<0.01) as compared to WT mice. There was a significant increase in TG2 crosslinks by IHC in WT old group compared to Young, with no stain in the TG2-/-animals. Optical microscopy examination of Old WT mice aorta showed thinning and fragmentation of elastic laminae. Young WT mice, old and young TG2-/-mice presented regularly arranged and parallel elastic laminae of the tunica media. CONCLUSION: The genetic suppression of TG2 delays the age-induced endothelial dysfunction and histological modifications.

Knockdown of transglutaminase-2 prevents early age-induced vascular changes in mice

Abstract Purpose: To determine whether the absence of transglutaminase 2 enzyme (TG2) in TG2 knockout mice (TG2-/-) protect them against early age-related functional and histological arterial changes. Methods: Pulse wave velocity (PWV) was measured using non-invasive Doppler and mean arterial pressure (MAP) was measured in awake mice using tail-cuff system. Thoracic aortas were excised for evaluation of endothelial dependent vasodilation (EDV) by wire myography, as well as histological analyses. Results: PWV and MAP were similar in TG2-/-mice to age-matched wild type (WT) control mice. Old WT mice exhibited a markedly attenuated EDV as compared to young WT animals. The TG2-/-young and old mice had enhanced EDV responses (p<0.01) as compared to WT mice. There was a significant increase in TG2 crosslinks by IHC in WT old group compared to Young, with no stain in the TG2-/-animals. Optical microscopy examination of Old WT mice aorta showed thinning and fragmentation of elastic laminae. Young WT mice, old and young TG2-/-mice presented regularly arranged and parallel elastic laminae of the tunica media. Conclusion: The genetic suppression of TG2 delays the age-induced endothelial dysfunction and histological modifications.

Nuclear transglutaminase 2 directly regulates expression of cathepsin S in rat cortical neurons.

Transglutaminase 2 (TG2) is a protein that modulates neuronal survival processes. Although TG2 is primarily cytosolic, data have suggested the nuclear localization of TG2 is strongly associated with neuronal viability. Depletion of TG2 in neurons results in neurite retraction and loss of viability, which is likely due to a dysregulation in gene expression. To begin to understand how TG2 regulates neuronal gene expression, chromatin immunoprecipitation was performed in neurons with TG2 overexpression. The resulting genomic DNA was recovered and sequenced. Bioinformatics analyses revealed that a signature DNA motif was enriched in the TG2 immunoprecipitated genomic DNA. In particular, this motif strongly mapped to a region proximate to the gene Ctss (cathepsin S). Knockdown of TG2 resulted in a significant increase in cathepsin S expression, which preceded the loss of neuronal viability. This is the first demonstration that TG2 directly associates with genomic DNA and regulates gene expression in neurons. Given that expression of cathepsin S is increased in neurological disease states, our data suggest that TG2 may play a role in promoting neuron health in part by repressing the expression of cathepsin S. Overall these data provide new insights into the function of nuclear TG2 in neurons.

Transglutaminases in autoimmune and inherited skin diseases: The phenomena of epitope spreading and functional compensation.

Transglutaminases (TGs) are structurally and functionally related enzymes that modify the post-translational structure and activity of proteins or peptides, and thus are able to turn on or switch off their function. Depending on location and activities, TGs are able to modify the signalling, the function and the fate of cells and extracellular connective tissues. Besides mouse models, human diseases enable us to appreciate the function of various TGs. In this study, skin diseases induced by genetic damages or autoimmune targeting of these enzymes will be discussed. TG1, TG3 and TG5 contribute to the cutaneous barrier and thus to the integrity and function of epidermis. TGM1 mutations related to autosomal recessive ichthyosis subtypes, TGM5 mutations to a mild epidermolysis bullosa phenotype and as novelty TGM3 mutation to uncombable hair syndrome will be discussed. Autoimmunity to TG2, TG3 and TG6 may develop in a few of those genetically determined individuals who lost tolerance to gluten, and manifest as coeliac disease, dermatitis herpetiformis or gluten-dependent neurological symptoms, respectively. These gluten responder diseases commonly occur in combination. In autoimmune diseases, the epitope spreading is remarkable, while in some inherited pathologies, a unique compensation of the lost enzyme function is noted.

Structural aspects of transglutaminase 2: functional, structural, and regulatory diversity.

Transglutaminase 2 (TG2) is a multi-functional protein that has both protein cross-linking and guanosine 5'-triphosphate (GTP) hydrolysis activities. The activities of this protein are controlled by many cellular factors, including calcium (Ca2+) and GTP, and have been implicated in several physiological activities, including apoptosis, angiogenesis, wound healing, cellular differentiation, neuronal regeneration, and bone development. TG2 is linked to many human diseases such as inflammatory disease, celiac disease, neurodegenerative disease, diabetes, tissue fibrosis, and various cancers and is one of the most dynamic enzymes in terms of its functions, structures, and regulatory mechanisms. The aim of this review was to summarize the functional, structural, and regulatory diversity of TG2, with a particular focus on the structure of TG2.

Receptor-stimulated transamidation induces activation of Rac1 and Cdc42 and the regulation of dendritic spines.

Regulation of dendritic spines is an important component of synaptic function and plasticity whereas dendritic spine dysregulation is related to several psychiatric and neurological diseases. In the present study, we tested the hypothesis that serotonin (5-HT)2A/2C receptor-induced Rho family transamidation and activation regulates dendritic spine morphology and that activation of multiple types of receptors can induce transglutaminase (TGase)-catalyzed transamidation of small G proteins. We previously reported a novel 5-HT2A receptor downstream effector, TGase-catalyzed serotonylation of the small G protein Rac1 in A1A1v cells, a rat embryonic cortical cell line. We now extend these findings to rat primary cortical cultures which develop dendritic spines; stimulation of 5-HT2A/2C receptors increased transamidation of Rac1 and Cdc42, but not RhoA. Inhibition of TGases significantly decreased transamidation and activation of Rac1 and Cdc42, suggesting that transamidation led to their activation. In primary cortical cultures, stimulation of 5-HT2A/2C receptors by 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) caused a transient dendritic spine enlargement, which was blocked by TGase inhibition. Stimulation of both 5-HT2A and 5-HT2C receptors contributed to DOI-induced Rac1 transamidation in primary cortical cultures as demonstrated by selective antagonists. Furthermore, stimulation of muscarinic acetylcholine receptors and NMDA receptors also increased TGase-catalyzed Rac1 activation in SH-SY5Y cells and N2a cells, respectively. Receptor-stimulated TGase-catalyzed transamidation of Rac1 occurs at Q61, a site previously reported to be important in the inactivation of Rac1. These studies demonstrate that TGase-catalyzed transamidation and activation of small G proteins results from stimulation of multiple types of receptors and this novel signaling pathway can regulate dendritic spine morphology and plasticity.

Deimination of Human Hornerin Enhances its Processing by Calpain-1 and its Cross-Linking by Transglutaminases.

Hornerin (HRNR) shares numerous features with filaggrin, a key contributor to the epidermal barrier functions. The two proteins display a related structural organization, are expressed by the granular keratinocytes as a large precursor processed by proteolysis, and are cross-linked to the cornified cell envelopes. Two main steps in the metabolism of filaggrin are its deimination and calpain-1 cleavage. Here, using ion-exchange chromatography and two-dimensional gel electrophoresis of human epidermis extracts, we determined that HRNR is deiminated in vivo. Accordingly, cornified envelopes, purified from plantar and abdominal human skin, were shown to contain deiminated proteins. A recombinant form of HRNR (HRNRHis) deiminated in vitro was shown to be a better substrate for transglutaminases 1 and 3 than the unmodified form. Our data also indicated that calpain-1 may be involved in the proteolytic processing of HRNR, because calpain-1 was co-located with HRNR in the cytoplasm of granular keratinocytes. Using Western blotting and mass spectrometry analysis, HRNRHis was shown to be cleaved by calpain-1 in vitro, its deimination enhancing its proteolysis. In HRNR full sequence, four calpain-1 cleavage sites were identified. Altogether, these data allowed a new role to be deciphered for deimination during cornification and provided further characterization of HRNR metabolism.

TG2 and NF-κB Signaling Coordinates the Survival of Mantle Cell Lymphoma Cells via IL6-Mediated Autophagy.

Expression of the transglutaminase TG2 has been linked to constitutive activation of NF-κB and chemotherapy resistance in mantle cell lymphoma (MCL) cells. TG2 forms complexes with NF-κB components, but mechanistic insights that could be used to leverage therapeutic responses has been lacking. In the current study, we address this issue with the discovery of an unexpected role for TG2 in triggering autophagy in drug-resistant MCL cells through induction of IL6. CRISPR-mediated silencing of TG2 delayed apoptosis while overexpressing TG2 enhanced tumor progression. Under stress, TG2 and IL6 mediate enhanced autophagy formation to promote MCL cell survival. Interestingly, the autophagy product ATG5 involved in autophagosome elongation positively regulated TG2/NF-κB/IL6 signaling, suggesting a positive feedback loop. Our results uncover an interconnected network of TG2/NF-κB and IL6/STAT3 signaling with autophagy regulation in MCL cells, the disruption of which may offer a promising therapeutic strategy. Cancer Res; 76(21); 6410-23. ©2016 AACR.