Coronary Artery Disease Associated Transcription Factor TCF21 Regulates Smooth Muscle Precursor Cells that Contribute to the Fibrous Cap.

TCF21 is a basic helix-loop-helix transcription factor that has recently been implicated as contributing to susceptibility to coronary heart disease based on genome wide association studies. In order to identify transcriptionally regulated target genes in a major disease relevant cell type, we performed siRNA knockdown of TCF21 in in vitro cultured human coronary artery smooth muscle cells and compared the transcriptome of siTCF21 versus siCONTROL treated cells. The raw (FASTQ) as well as processed (BED) data from 3 technical replicates per treatment has been deposited with Gene Expression Omnibus (GSE44461).

Osteoprotegerin, a crucial regulator of bone metabolism, also regulates B cell development and function.

Osteoprotegerin (OPG) is a CD40-regulated gene in B cells and dendritic cells (DCs). We investigated the role of OPG in the immune system by generating opg(-/-) mice. Like its role as a regulator of bone metabolism, OPG also influences processes in the immune system, notably in B cell development. Ex vivo, opg(-/-) pro-B cells have enhanced proliferation to IL-7, and in opg(-/-) spleen, there is an accumulation of type 1 transitional B cells. Furthermore, opg(-/-) bone marrow-derived DCs are more effective in stimulating allogeneic T cells than control DCs. When challenged with a T-dependent Ag, opg(-/-) mice had a compromised ability to sustain an IgG3 Ag-specific response. Thus, in the immune system, OPG regulates B cell maturation and development of efficient Ab responses.

Retention of PDGFR-beta function in mice in the absence of phosphatidylinositol 3'-kinase and phospholipase Cgamma signaling pathways.

Signal transduction by the platelet-derived growth-factor receptor beta (PDGFR-beta) tyrosine kinase is required for proper formation of vascular smooth muscle cells (VSMC). However, the importance of individual PDGFR-beta signal transduction pathways in vivo is not known. To investigate the role of two of the pathways believed to be critical for PDGF signal transduction, we have generated mice that bear a PDGFR-beta that can no longer activate PI3kinase or PLCgamma. Although these mutant mice have normal vasculature, we provide multiple lines of evidence in vivo and from cells derived from the mutant mice that suggest that the mutant PDGFR-beta operates at suboptimal levels. Our observations indicate that although loss of these pathways can lead to attenuated PDGF-dependent cellular function, certain PDGFR-beta-induced signal cascades are not essential for survival in mice.

Early myotome specification regulates PDGFA expression and axial skeleton development.

Reciprocal defects in signaling between the myotome and the sclerotome compartments of the somites in PDGFRalpha and Myf5 mutant embryos lead to alterations in the formation of the vertebrae and the ribs. To investigate the significance of these observations, we have examined the role of PDGF signaling in the developing somite. PDGFA ligand expression was not detected in the myotome of Myf5 null mutant embryos and PDGFA promoter activity was regulated by Myf5 in vitro. PDGFA stimulated chondrogenesis in somite micromass cultures as well as in embryos when PDGFA was knocked into the Myf5 locus, resulting in increased vertebral and rib development. PDGFA expression in the myotome was fully restored in embryos in which MyoD has been introduced at the Myf5 locus but to a lesser extent in similar myogenin knock-in embryos. These results underscore the importance of growth factor signaling within the developing somite and suggest an important role for myogenic determination factors in orchestrating normal development of the axial skeleton.

T cell receptor interactions with class I heavy-chain influence T cell selection.

The interaction of the T cell receptor (TCR) with peptide in the binding site of the major histocompatibility complex molecule provides the basis for T cell recognition during immune surveillance, repertoire development, and tolerance. Little is known about the extent to which repertoire selection is influenced directly by variation of the structure of the class I heavy chain. We find that the 2C TCR, normally positively selected in the context of the K(b) molecule, is minimally selected into the CD8 lineage in the absence of antigen-processing genes. This finding underscores the importance of peptides in determining the positive-selecting class I ligands in the thymus. In contrast, K(bm3), a variant class I molecule that normally exerts a negative selection pressure on 2C-bearing T cells, positively selects 2C transgenic T cells into the CD8 lineage in an antigen-processing gene-deficient environment. These findings indicate that structural changes in the heavy chain can have direct influence in T cell recognition, from which we conclude that the nature of TCR interaction with class I heavy chain influences the array of TCRs selected during development of the functional adult repertoire.

Growth factor signaling pathways in vascular development.

Recent research on the formation and maintenance of the vasculature in the embryo and in the adult has provided a greater understanding of the cellular signals involved in these processes. With this understanding comes the potential means of controlling vascularization in pathological situations such as tumorigenesis and wounding. For the purpose of this review, we will discuss the key receptor tyrosine kinases involved in vascular function and the molecules which relay signals downstream of receptor activation. The receptor tyrosine kinases discussed include the vascular endothelial cell growth factor receptors, Eph receptors, Tie1, and Tie2, all of which are expressed on vascular endothelial cells. We also discuss the roles of the platelet derived growth factor receptors which are expressed on vascular smooth muscle cells. While all of these receptor tyrosine kinases activate many similar effector molecules, some of the signals initiated appear to be distinct. This may explain, at least in part, how different receptor tyrosine kinases expressed in overlapping patterns on the developing vasculature, direct unique biological functions.

Degenerate recognition of alloantigenic peptides on a positive-selecting class I molecule.

The well-defined 2C T cell was used to investigate alloreactive degeneracy. A panel of class I molecules that are known ligands for the 2C TCR were sensitized with three known peptide ligands, p2Ca (LSPFPFDL), dEV-8 (EQYKFYSV), and SIYR-8 (SIYRYYGL). The peptide p2Ca was originally identified as the allopeptide seen in the Ld class I molecule by 2C T cells, 2C recognizes the dEV-8 peptide as the ligand in the Kbm3 class I molecule, and SIYR-8 was recently identified as a peptide ligand for 2C in the context of the Kb class I molecule. Strong recognition of all three Ag-presenting molecules occurred in the context of their respective allopeptides, but 2C recognized all three peptides to a measurable extent in the context of Kb. Molecular modeling of these Kb/peptide complexes revealed a high degree of similarity between dEV-8 and SIYR-8, but very little conformational similarity of either of these peptides with p2Ca. Furthermore, the structural changes in the mutant Kbm3 binding site resulted in generalized changes in the conformation of each of five bound peptides compared with those of the same peptides bound to Kb. The finding that degenerate recognition occurs on Kb, the restriction element responsible for selecting 2C T cells, suggests a unique relationship between a TCR and the Ag-presenting molecule that mediates its positive selection.

Alphabeta T cell receptor interactions with syngeneic and allogeneic ligands: affinity measurements and crystallization.

Cellular immunity is mediated by the interaction of an alphabeta T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have alphabeta TCRs that can recognize both self- and foreign peptide-MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive alphabeta T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired alphabeta heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2Kb-dEV8, H-2Kbm3-dEV8, H-2Kb-SIYR, and H-2Ld-p2Ca spans a range of affinities from Kd = 10(-4) to 10(-6)M for the syngeneic (H-2Kb) and allogeneic (H-2Kbm3, H-2Ld) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the alpha and beta chains. X-ray quality crystals of complexes of 2C with H-2Kb-dEV8, H-2Kbm3-dEV8 and H-2Kb-SIYR have been grown.

A single T cell receptor recognizes structurally distinct MHC/peptide complexes with high specificity.

The 2C T cell is a CD8+, alloreactive T cell, which recognizes cells bearing Ld and Kbm3 class I major histocompatability complex molecules. Here, we characterize an allopeptide, designated dEV-8, that is a ligand in the Kbm3 molecule for the 2C TCR but is not a ligand in the Ld molecule. By biochemical and immunological properties, dEV-8 is distinct from P2Ca, the Ld allopeptide that is also recognized by the 2C TCR. Using the deduced amino acid sequence of dEV-8, we isolate a candidate endogenous source of the peptide. The endogenous protein, MLRQ, contains a peptide sequence identical to dEV-8. This degenerate recognition of two distinct peptide/MHC complexes by a single TCR has important implications for understanding allorecognition.

Alloreactive 2C T cells recognize a self peptide in the context of the mutant Kbm3 molecule.

Previous studies hav shown that 2C T cells have dual specificity for Ld and Kbm3 alloantigens and are positively selected in the context of Kb. Analysis of Kbm3-eluted peptides reveals a single HPLC fraction that is a central component of the Kbm3 allodeterminant. Peptides with biologic and biochemical properties indistinguishable from those of the Kbm3 allopeptide were also isolated from Kb and Kbm8, despite the fact that cells bearing these class I molecules are not efficient targets for 2C T cells. The allopeptide was isolated and compared with p2Ca, the Ld allopeptide recognized by 2C T cells in the context of Ld. The Kbm3 allopeptide was found to be biochemically and functionally different from p2Ca. Therefore, the 2C TCR has dual specificity for two different peptides presented in the context of two structurally distinct class I molecules. The possibility of a common peptide being bound by Kb, Kbm3, and Kbm8 suggests that the peptide defined as an allodeterminant in the context of Kbm3 may also function in the positive selection of 2C T cells when presented in the context of Kb and Kbm8 during thymic development. In support of this view, the allopeptide was also found in Kb and Kbm8 thymic peptide eluates.

Minor pocket B influences peptide binding, peptide presentation and alloantigenicity of H-2Kb.

Microsequence analysis of peptides eluted from the murine class I H-2Kb molecule together with the three-dimensional structure of the molecule co-crystallized with a homogeneous population of peptides suggests that pocket B is a minor pocket that does not play a major role in peptide presentation. This is in contrast to most other class I molecules in which pocket B plays a central role in selecting and presenting antigenic peptides. To investigate the role of pocket B in antigen presentation by the Kb molecule, we analyzed site-directed mutants of position 45 in pocket B for their effect on both allo- and peptide-specific recognition. We made an identical set of mutations in Kbm8 at residue 45 in order to evaluate their influence in the context of a more open pocket B which results from the bm8 substitution at amino acid 24 (E-->S). We demonstrated that this minor pocket did play a significant role in the antigenicity of both molecules and that this role was more readily apparent in the context of the more open pocket B of Kbm8. In addition, we found that some substitutions of residue 45 in the Kbm8 molecule restored recognition by some alloreactive and peptide specific anti-Kb T cell clones which are normally restricted to Kb, indicating that multiple configurations of amino acids in a pocket could result in similar binding and presentation capabilities.

Recognition of a single amino acid change on the surface of a major transplantation antigen is in the context of self peptide.

The transcripts encoding two strongly alloantigenic class I mutant molecules, Kdm4 and Kdm5, were characterized and found to encode products that differ from the parental Kd glycoprotein by single amino acid substitutions. The Kdm4 molecule has an amino acid change at position 114, an integral component of a beta-sheet associated with pockets D and E of the peptide binding site. The basis for strong alloantigenicity of the variant molecule can be attributed to differences in peptide binding that were visualized by HPLC analysis of eluted peptides. In contrast, the Kdm5 molecule differs from the parent at position 158, a component of the alpha-helix that is not associated with any of the pockets of the peptide binding site. No differences in peptide binding by Kdm5 in comparison with the parent Kd molecule were seen by HPLC, suggesting that the variant and parent molecules bind the same set of peptides. The ability of (dm4 x dm5) F1 hybrid mice to recognize and lyse BALB/c stimulator cells indicates that the alloantigenic properties determined by the 158 substitution result from the interactions of the alpha-helix regions (changed in dm5) with the pockets of the binding site (changed in dm4). We conclude that self peptides shared by the F1 hybrid and the BALB/c stimulator cells are recognized in the context of structural features of the helices of the Ag-presenting molecule as alloantigenic determinants.