Effects of calreticulin mutations on cell transformation and immunity.

Myeloproliferative neoplasms (MPNs) are cancers involving dysregulated production and function of myeloid lineage hematopoietic cells. Among MPNs, Essential thrombocythemia (ET), Polycythemia Vera (PV) and Myelofibrosis (MF), are driven by mutations that activate the JAK-STAT signalling pathway. Somatic mutations of calreticulin (CRT), an endoplasmic reticulum (ER)-localized lectin chaperone, are driver mutations in approximately 25% of ET and 35% of MF patients. The MPN-linked mutant CRT proteins have novel frameshifted carboxy-domain sequences and lack an ER retention motif, resulting in their secretion. Wild type CRT is a regulator of ER calcium homeostasis and plays a key role in the assembly of major histocompatibility complex (MHC) class I molecules, which are the ligands for antigen receptors of CD8+ T cells. Mutant CRT-linked oncogenesis results from the dysregulation of calcium signalling in cells and the formation of stable complexes of mutant CRT with myeloproliferative leukemia (MPL) protein, followed by downstream activation of the JAK-STAT signalling pathway. The intricate participation of CRT in ER protein folding, calcium homeostasis and immunity suggests the involvement of multiple mechanisms of mutant CRT-linked oncogenesis. In this review, we highlight recent findings related to the role of MPN-linked CRT mutations in the dysregulation of calcium homeostasis, MPL activation and immunity.

Activation of αvß3 Integrin Alters Fibronectin Fibril Formation in Human Trabecular Meshwork Cells in a ROCK-Independent Manner.

Purpose: Fibronectin fibrillogenesis is an integrin-mediated process that may contribute to the pathogenesis of primary open-angle glaucoma (POAG). Here, we examined the effects of αvß3 integrins on fibrillogenesis in immortalized TM-1 cells and human trabecular meshwork (HTM) cells. Methods: TM-1 cells overexpressing wild-type ß3 (WTß3) or constitutively active ß3 (CAß3) integrin subunits were generated. Control cells were transduced with an empty vector (EV). Deoxycholic acid (DOC) extraction of monolayers, immunofluorescence microscopy, and On-cell western analyses were used to determine levels of fibronectin fibrillogenesis and fibronectin fibril composition (EDA+ and EDB+ fibronectins) and conformation. αvß3 and α5ß1 Integrin levels were determined using fluorescence-activated cell sorting (FACS). Cilengitide and an adenovirus vector expressing WTß3 or CAß3 integrin subunits were used to examine the role of αvß3 integrin in HTM cells. The role of the canonical α5ß1 integrin-mediated pathway in fibrillogenesis was determined using the fibronectin-binding peptide FUD, the ß1 integrin function-blocking antibody 13, and the Rho kinase (ROCK) inhibitor Y27632. Results: Activation of αvß3 integrin enhanced the assembly of fibronectin into DOC-insoluble fibrils in both TM-1 and HTM cells. The formation of fibronectin fibrils was dependent on α5ß1 integrin and could be inhibited by FUD. However, fibrillogenesis was unaffected by Y27632. Fibrils assembled by CAß3 cells also contained high levels of EDA+ and EDB+ fibronectin and fibronectin that was stretched. Conclusions: αvß3 Integrin signaling altered the deposition and structure of fibronectin fibrils using a ß1 integrin/ROCK-independent mechanism. Thus, αvß3 integrins could play a significant role in altering the function of fibronectin matrices in POAG.

Genomic/proteomic analyses of dexamethasone-treated human trabecular meshwork cells reveal a role for GULP1 and ABR in phagocytosis.

Purpose: The purpose of this study is to examine the expression profile of genes related to integrin-mediated phagocytosis that are altered by dexamethasone (DEX) and/or αvß3 integrin signaling to gain a better understanding of the molecular basis of phagocytosis and the pathophysiology of glucocorticoid-induced ocular hypertension. Methods: RNA and cell lysates were obtained from human trabecular meshwork (HTM) cells incubated with and without DEX for 4-5 d. The relative level of gene expression was evaluated using the Affymetrix Gene Chip® human gene microarray and quantitative PCR (qPCR). Changes in protein expression were validated using western blots or FACS analyses. The involvement of proteins in phagocytosis was determined using siRNA to knock down the expression of these proteins in an immortalized TM-1 cell line. Changes in the phagocytic activity were measured using pHrodo™-labeled S. aureus bioparticles followed by immunofluorescence microscopy. The effect of αvß3 integrin expression and activity on GULP1 mRNA levels was measured using qPCR in TM-1 cells overexpressing wild type or constitutively active αvß3 integrin. Results: Gene microarrays revealed statistically significant differences (>2 fold) in the expression of seven genes known to be involved in phagocytosis. Three genes (CD36, ABR, and GULP1) were downregulated, while four genes (ITGB3, CHN1, PIK3R1, and MFGE8) were upregulated. The genes were either associated with modulating RAC1 activity (ABR and CHN1) or integrin signaling (CD36, GULP1, ITGB3, PIK3R1, and MFGE8). Another gene, SIRPA, was also downregulated (1.6 fold) but only in one cell strain. qPCR and western blot analyses verified that DEX caused a decrease in SIRPA and GULP1 mRNA and their protein levels, while levels of CHN1 mRNA and its protein were upregulated by DEX. qPCR showed that although ABR mRNA was downregulated compared to non-treated controls after 5 d of treatment with DEX, no change at the protein level was detected. qPCR analysis also revealed that DEX caused an increase in MFGE8 mRNA levels. The levels of CD36 mRNA and protein varied between cell strains treated with DEX and were not statistically different compared to controls. The knockdown of GULP1 and ABR using siRNAs decreased phagocytosis by 40%. Interestingly, GULP1 mRNA levels were also decreased by 60% when αvß3 integrin was overexpressed in TM-1 cells. Conclusion: The DEX-induced inhibition of phagocytosis may be caused by the downregulation of ABR and GULP1 disrupting the αvß5 integrin/RAC1-mediated engulfment pathway. The downregulation of GULP1 by αvß3 integrin further suggests that this integrin may be a negative regulator of phagocytosis by transcriptionally downregulating proteins needed for phagocytosis. In summary, these results represent new insights into the effects of glucocorticoids and integrin signaling on the phagocytic process in the TM.