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In this issue of Arteriosclerosis, Thrombosis and Vascular Biology, Westmuckett and Moore describe a prominent role of tyrosine sulfation in hematopoietic cells during atherosclerosis development 1. Atherosclerosis represents a major health problem in developed and developing countries and is the most common underlying process precipitating myocardial infarctions, limb ischemia and stroke. In the United States alone, over 60 million people have some form of atherosclerotic vascular disease 2. Atherosclerosis is a complex disease associated with lipid accumulation and formation of atherosclerotic plaque in the major arteries of the body, which can rupture or erode to cause acute thrombosis, arterial occlusion and necrosis of the dependent tissue.
In a last two decades, it has become clear that immune processes play an important role in atherosclerosis development, plaque formation and progression of the disease. Although all major types of immune cells are involved in atherosclerosis (reviewed in 3), the exact molecular and cellular mechanisms of disease development and progression are not completely understood. Two commonly used mouse models of atherosclerosis are apolipoprotein E-deficient Apoe−/− or low density lipoprotein (LDL) receptor-deficient Ldlr−/− mice. In both models, LDL cholesterol is elevated, which promotes atherosclerotic lesion formation in locations similar to those in the human disease. Ldlr−/− mice can be reconstituted with bone marrow from donor mice to produce chimeric mice lacking targeted molecules of interest in hematopoietic cells only.
Leukocyte recruitment is critical for chronic inflammation and progression of atherosclerosis. This process is regulated by adhesion molecules and chemoattractants such as chemokines and their receptors 4. One class of adhesion molecules required for recruitment of inflammatory the cells to sites of atherosclerotic lesion formation are selectins. P-E-and L-select in all can bind P-select in glycoprotein ligand-1 (PSGL-1) 5.
The expression and function of adhesion molecules and chemokine receptors is controlled at the transcriptional and translational levels as well as by posttranslational modifications. Many intracellular proteins are regulated by phosphorylation, but other types of covalent modifications exist. Sulfation of tyrosine residues turns out to be critical for the function of many secreted and transmembrane proteins, which are involved in both homeostasis and immune responses 6. Two broadly expressed enzymes catalyzing the transfer of the sulfoil group from 3′-phosphoadenosin 5′-phosphosulfate(PAPS) to the hydroxyl group of peptidyl-tyrosine within these proteins have been described in humans and mice, the tyrosylprotein sulfotransferases-1 and -2 (TPST-1, TPST-2). Disruption of Tpst genes by gene targeting and homologous recombination in mice demonstrates important roles for these enzymes in crucial biological functions, such as metabolism, reproduction and cardiovascular development. For instance, Tpst1−/− mice have a significant decrease of postnatal body weight due to impaired digestion 7. Male Tpst2−/− mice have reduced fertility due to impaired sulfation of proteins responsible for male reproductive function 8. Although at least some Tpst1−/− Tpst2−/− double knockout animals were born alive, most of them died due to cardiopulmonary deficiency in the early postnatal period9.
Previous work has shown that sulfation of tyrosine residues in N-terminal regions of chemokine receptors plays an important role in interaction with their chemokine ligands. Intriguingly, O-sulfation was observed in a number of chemokine receptors. Some of these are implicated in atherosclerosis progression, including the chemokine receptors CCR2, CCR5, CX3CR1. Selective ablation of CCR210, CCR511 or CX3CR112, 13 significantly decreases lesion formation in atherosclerosis-prone mice, likely due to impaired recruitment of mononuclear cells.
To study a functional role of tyrosine sulfation in chemokine receptors, several groups introduced point mutations to substitute Phe for Tyr. For example, cells bearing point mutations in tyrosine residues in CXCR3 were unable to migrate effectively toward the CXCR3 ligands CXCL9, CXCL10 and CXCL1114. Mutation in one of two N-terminal tyrosine residues of CX3CR1 caused a 100-fold decrease of affinity for its ligand CX3CL1, whereas mutation in another tyrosine residue (Tyr 22) decreased cell adhesion15. The function of both CCR5 and CXCR4 was also modified by tyrosine sulfation 16, 17 as shown by reduced migration to their cognate ligands when tyrosines were mutated to phenylalanines.
Tyrosine sulfation is also critical for PSGL-1 function, since mutation in N-terminal tyrosine residues dramatically reduced the affinity of PSGL-1 for P-selectin18, 19. Many of these studies addressing the role of tyrosine sulfation for the interaction of ligand-receptor pairs were performed in vitro, and the exact relevance of this process in vivo remained unclear. The global pathophysiological role of tyrosine sulfation in atherosclerosis has never been examined.
A study published in this issue 1 uncovers an important role for tyrosine sulfation in the atherosclerosis development. The authors generated bone marrow chimeric mice using hematopoietic progenitors from tyrosylprotein sulfotransferase deficient animals lacking both TPST-1 and -2 and studied the Tpst1−/− Tpst2−/− mutation in hematopoietic cells in the atherosclerosis-prone Ldlr−/− genetic background. TSPT deficient (Tpst1−/− Tpst2−/− -> Ldlr−/−) and control (WT-> Ldlr−/−) mice were fed a high-fat atherogenic diet and atherosclerosis development was analyzed. The authors found significantly reduced atherosclerotic plaques in mice with defective tyrosine sulfation in hematopoietic cells. Importantly, necrotic areas and macrophage numbers were significantly decreased in these mice compared with the control group. The authors also showed that murine PSGL-1 and CX3CR1 are tyrosine sulfated(figure 1). Tyrosine sulfation is completely absent in Tpst1−/− Tpst2−/− mice, and therefore PSGL-1 and CX3CR1 are likely non-functional in the absence of sulfation in bone marrow chimeric mice (Tpst1−/− Tpst2−/− -> Ldlr−/−). Thus, their studies clearly establish that tyrosine sulfation in bone marrow-derived cells play a critical role in atherosclerotic plaque formation and growth. However, it remains unclear what type of hematopoietic cells play a major role in connecting tyrosine sulfation and atherosclerosis. It is tempting to speculate that both macrophages and T cells may be affected by the lack of tyrosine sulfation, since these cell types are the most important immune subsets enriched in the plaque. Although tyrosine sulfation was described for CCR2, CCR5 and CX3CR1, chemokine receptors involved in recruitment of inflammatory cells into atherosclerosis plaque, it remains to be investigated whether tyrosine sulfation of these (or possibly other) chemokine receptors plays a predominant role in atherosclerosis development. The role of PSGL-1 tyrosine sulfation also remains to be investigated in detail. From the Tpst1−/− Tpst2−/− mice described in 1, leukocytes could be isolated and tested in flow chambers for their ability to tether and roll on P-selectin, which is likely the PSGL-1 function most relevant for atherosclerosis.
Tyrosylprotein sulfotransferases might also be a target for pharmacologic intervention to prevent or treat atherosclerosis or other inflammatory diseases. Potential side effects must be expected due to the broad expression of these enzymes. Tyrosylprotein sulfotransferases may be inhibitable by small molecules targeting their catalytic center. Development of specific antibodies, which could distinguish between sulfated and non-sulfated proteins in vivo, may be another potential direction for creating an anti-atherosclerosis therapy based on limiting immune cell recruitment into the area of inflammation. To be successful, the key receptors that require tyrosine sulfation and are indispensible for atherosclerosis development must be determined. The study by Westmuckett and Moore 1 suggests that such targets exist and can be found.