Monocyte Adhesion and Plaque Recruitment During Atherosclerosis Development Is Regulated by the Adapter Protein Chat-H/SHEP1.

OBJECTIVE: The chronic inflammation associated with atherosclerosis is caused by lipid deposition followed by leukocyte recruitment to the arterial wall. We previously showed that the hematopoietic cell-specific adaptor protein Cas- and Hef1-associated signal transducer hematopoietic isoform (Chat-H)/SHEP1 regulated lymphocyte adhesion and migration. In this study, we analyzed the role of Chat-H in atherosclerosis development. APPROACH AND RESULTS: Using Chat-H-deficient bone marrow transplantation in low-density lipoprotein receptor-deficient mice, we found that Chat-H regulated atherosclerotic plaque formation. Chat-H deficiency in hematopoietic cells associated with lower plaque complexity and fewer leukocytes in the lesions, whereas myeloid-specific deletion of Chat-H was sufficient for conferring atheroprotection. Chat-H deficiency resulted in reduced recruitment of classical Ly6c(high) and nonclassical Ly6c(low) monocytes to the plaques, which was accompanied by increased numbers of both monocyte subsets in the blood. This associated with defective adhesion of Chat-H-deficient Ly6c(high) and Ly6c(low) monocytes to vascular cell adhesion molecule-1 in vitro and impaired infiltration of fluorescent bead-loaded monocytes to atherosclerotic plaques. In contrast, Chat-H was dispensable for CX3CL1 and CCR1/CCR5-dependent migration of monocytes. CONCLUSIONS: Our findings highlight Chat-H as a key protein that regulates atherosclerosis development by controlling monocyte adhesion and recruitment to the plaques and identify a novel target that may be exploited for treating atherosclerosis.

Regulation of T-lymphocyte physiology by the Chat-H/CasL adapter complex.

The Cas family of proteins consists of at least four members implicated in the regulation of diverse cellular processes such as cell proliferation, adhesion, motility, and cancer cell metastasis. Cas family members have conserved C-termini that mediate constitutive heterotypic interactions with members of a different group of proteins, the NSP family. Both the Cas and NSP proteins have conserved domains that mediate protein-protein interactions with other cytoplasmic intermediates. Signaling modules assembled by these proteins in turn regulate signal transduction downstream of a variety of receptors including integrin, chemokine, and antigen receptors. T lymphocytes express the NSP protein NSP3/Chat-H and the Cas protein Hef1/CasL, which are found in a constitutive complex in naive T cells. We recently showed that Chat-H and Hef1/CasL regulate integrin-mediated adhesion and promote T-cell migration and trafficking downstream of activated chemokine receptors. It is currently unclear if the Chat-H/CasL module also plays a role in antigen receptor signaling. Here we review our current knowledge of how Chat-H and Hef1/CasL regulate T-cell physiology and whether this protein complex plays a functional role downstream of T-cell receptor activation.

The hematopoietic isoform of Cas-Hef1-associated signal transducer regulates chemokine-induced inside-out signaling and T cell trafficking.

Leukocyte migration and trafficking is dynamically regulated by various chemokine and adhesion molecules and is vital to the proper function of the immune system. We describe a role for the Cas and Hef-1-associated signal transducer in hematopoietic cells (Chat-H) as a critical regulator of T lymphocyte migration, by using lentivirus-mediated RNA interference (RNAi). Impaired migration of Chat-H-depleted cells coincided with defective inside-out signaling shown by diminished chemokine-induced activation of the Rap-1 GTPase and integrin-mediated adhesion. Localization of Chat-H to the plasma membrane, association with its binding partner Crk-associated substrate in lymphocytes (CasL), and Chat-H-mediated CasL serine-threonine phosphorylation were required for T cell migration. These results identify Chat-H as a critical signaling intermediate acting upstream of Rap1 to regulate chemokine-induced adhesion and migration.

Sin: good or bad? A T lymphocyte perspective.

Stimulation of T cells through their antigen receptor induces a multitude of signaling networks that regulate T cell activation in the form of cytokine production and T cell proliferation. Multiple signal integration sites exist along these pathways in the form of multiprotein signaling complexes, the formation of which is facilitated by adapter and scaffold molecules. In recent years a number of adapter and scaffold molecules have been described in T cells and shown to play an integral part in T cell function. Among these molecules are proteins that function as positive or negative regulators of T cell activation downstream of the activated T cell receptor (TCR). Here, we discuss the role of a small family of multiadapter proteins on T cell activation, the p130Cas family, with emphasis on one of its members, Sin (Src-interacting protein). Our results suggest that Sin inhibits thymocyte development and T cell activation and is a novel negative regulator of T lymphocyte function.

Mutational analysis of the Escherichia coli PhoQ sensor kinase: differences with the Salmonella enterica serovar Typhimurium PhoQ protein and in the mechanism of Mg2+ and Ca2+ sensing.

The PhoP-PhoQ two-component system plays a role in Mg2+ homeostasis and/or the virulence properties of a number of bacterial species. A Salmonella enterica serovar Typhimurium PhoQ sensor kinase mutant, in which the threonine at residue 48 in the periplasmic sensor domain is changed to an isoleucine, was shown previously to result in elevated expression of PhoP-activated genes and to affect mouse virulence, epithelial cell invasion, and sensitivity to macrophage killing. We characterized a complete set of proteins having amino acid substitutions at position 48 in the closely related Escherichia coli PhoQ protein. Numerous mutant proteins having amino acid substitutions with side chains of various sizes and characters displayed signaling phenotypes similar to that of the wild-type protein, indicating that interactions mediated by the wild-type threonine side chain are not required for normal protein function. Changes to amino acids with aromatic side chains had little impact on signaling in response to extracellular Mg2+ but resulted in reduced sensitivity to extracellular Ca2+, suggesting that the mechanisms of signal transduction in response to these two divalent cations are different. Surprisingly, the Ile48 protein displayed a defective phenotype rather than the hyperactive phenotype seen with the S. enterica serovar Typhimurium protein. We also describe a mutant PhoQ protein lacking the extracellular sensor domain with a defect in the ability to activate PhoP. The defect does not appear to be due to reduced autokinase activity but rather appears to be due to an effect on the stability of the aspartyl-phosphate bond of phospho-PhoP.

Defective thymocyte maturation by transgenic expression of a truncated form of the T lymphocyte adapter molecule and Fyn substrate, Sin.

Adapter molecules that promote protein-protein interactions play a central role in T lymphocyte differentiation and activation. In this study, we examined the role of the T lymphocyte-expressed adapter protein and Src kinase substrate, Sin, on thymocyte function using transgenic mice expressing an activated, truncated allele of Sin (SinDeltaC). We found that SinDeltaC expression led to reduced numbers of CD4(+) and CD8(+) single-positive cells and reduced thymic cellularity due to increased thymocyte apoptosis. Because the adapter properties of Sin are mediated by tyrosine-based motifs and given that Sin is a substrate for Src tyrosine kinases, we examined the involvement of these kinases in the inhibitory effects of SinDeltaC. We found that in transgenic thymocytes, SinDeltaC was constitutively phosphorylated by the Src kinase Fyn, but not by the related kinase Lck. Using SinDeltaC and fyn(-/-) animals, we also found that the expression of Fyn was required for the inhibitory effect of SinDeltaC on thymocyte apoptosis but not for SinDeltaC-mediated inhibition of T cell maturation. The inhibitory effect of SinDeltaC on thymocyte maturation correlated with defective activation of the mitogen-activated protein kinase extracellular signal-regulated kinase. Our results suggest that the Sin mutant inhibits thymocyte differentiation through Fyn-dependent and -independent mechanisms and that endogenous Sin may be an important regulator of thymocyte development.