Cloning of a novel retinoid-inducible serine carboxypeptidase from vascular smooth muscle cells.

Retinoids block smooth muscle cell (SMC) proliferation and attenuate neointimal formation after vascular injury, presumably through retinoid receptor-mediated changes in gene expression. To identify target genes in SMC whose encoded proteins could contribute to such favorable biological effects, we performed a subtractive screen for retinoid-inducible genes in cultured SMC. Here, we report on the cloning and initial characterization of a novel retinoid-inducible serine carboxypeptidase (RISC). Expression of RISC is low in cultured SMC but progressively increases over a 5-day time-course treatment with all-trans-retinoic acid. A near full-length rat RISC cDNA was cloned and found to have a 452-amino acid open reading frame containing an amino-terminal signal sequence, followed by several conserved domains comprising the catalytic triad common to members of the serine carboxypeptidase family. In vitro transcription and translation experiments showed that the rat RISC cDNA generates an approximately 51-kDa protein. Confocal immunofluorescence microscopy of COS-7 cells transiently transfected with a RISC-His tag plasmid revealed cytosolic localization of the fusion protein. Western blotting studies using conditioned medium from transfected COS-7 cells suggest that RISC is a secreted protein. Tissue Northern blotting studies demonstrated robust expression of RISC in rat aorta, bladder, and kidney with much lower levels in all other tissues analyzed; high level RISC expression was also observed in human kidney. In situ hybridization verified the localization of RISC to medial SMC of the adult rat aorta. Interestingly, expression in kidney was restricted to proximal convoluted tubules; little or no expression was observed in glomerular cells, distal convoluted and collecting tubules, or medullary cells. Radiation hybrid mapping studies placed the rat RISC locus on chromosome 10q. These studies reveal a novel retinoid-inducible protease whose activity may be involved in vascular wall and kidney homeostasis.

Subtractive hybridization reveals tissue-specific expression of ahnak during embryonic development.

The gene product ahnak has been identified from extra-embryonic mesoderm cDNA enriched using a subtractive hybridization approach modified for using small amounts of starting material. Clones for cyclin D2 and H19 have also been isolated as being preferentially enriched in the extra-embryonic mesoderm compared with the embryo proper of embryonic day (E) 7.5 neural plate stage mouse embryos. The differential expression of these genes was confirmed at gastrulation stage using in situ hybridization. More detailed analysis of the human genomic ahnak sequence suggests that its highly repetitive structure was formed by unequal cross-over and gene conversion. During organogenesis, ahnak is expressed in a variety of tissues, including migratory mesenchyme. By E12.5, the major site of expression of ahnak is craniofacial mesenchyme. Immunohistochemical analysis has shown that ahnak protein is expressed mainly at the cell membrane of migratory mesenchymal cells, primarily in the nucleus of bone growth plate cells and mostly in the cytoplasm of differentiating nasal epithelia. The potential functions of ahnak are discussed in light of these results.

A novel retinoid-response gene set in vascular smooth muscle cells.

A modified suppression subtractive hybridization assay was performed to uncover genes induced by all-trans retinoic acid in cultured smooth muscle cells (SMC). Northern blotting studies confirmed the induction of 14 genes, many of which have heretofore been unrecognized as retinoid-inducible. Temporal expression and cycloheximide studies allowed us to categorize these genes as either immediate-early (LOX-1, endolyn, Stoned B/TFIIA alpha/beta-like factor, Src Suppressed C Kinase Substrate, and tissue transglutaminase) or delayed (cathepsin-L, ceruloplasmin, epithelin, importin alpha, alpha(8)-integrin, lactate dehydrogenase B, retinol dehydrogenase, spermidine/spermine N(1)-acetyltransferase, and VCAM-1) retinoid-response genes. A survey of rat tissues showed two of the genes (tissue transglutaminase and alpha(8)-integrin) to be highly restricted to vascular tissue. In situ hybridization verified expression of both tissue transglutaminase and alpha(8)-integrin to SMC in balloon-injured rat carotid artery. These findings unveil a new retinoid-response gene set that should be exploited to define molecular pathways involved in the antagonistic effects of retinoids on SMC growth and neointimal formation.

Diverse spatial expression patterns of UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase family member mRNAs during mouse development.

Cell migration and adhesion during embryonic development are complex processes which likely involve interactions among cell-surface carbohydrates. While considerable work has implicated proteoglycans in a wide range of developmental events, only limited attention has been directed towards understanding the 7role(s) played by the related class of mucin-type O-glycans. The initial step of mammalian mucin-type O-glycosylation is catalyzed by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases). The spatial expression patterns of the messenger RNAs of seven ppGaNTase family members were investigated from gastrulation through organogenesis stages of mouse development. The seven glycosyltransferases were expressed in unique patterns during embryogenesis. ppGaNTase-T1, -T2, -T4, and -T9 were expressed more ubiquitously than ppGaNTase-T3, -T5, and -T7. Organ systems with discrete accumulation patterns of ppGaNTase family members include the gastrointestinal tract (intestine, liver, stomach, submandibular gland), nervous system (brain, eye), lung, bone, yolk sac, and developing craniofacial region. The pattern in the craniofacial region included differential expression by family members in developing mandible, teeth, tongue and discrete regions of the brain including the pons and migratory, differentiating neurons. Additionally, ppGaNTase-T5 accumulates in a subset of mesenchymal cells at the ventral-most portions of the E12.5 maxilla and mandible underlying the dental lamina. The unique spatiotemporal expression of the different ppGaNTase family members during development suggests unique roles for each of these gene products.

Embryonic expression and function of the chemokine SDF-1 and its receptor, CXCR4.

Directed cell movement is integral to both embryogenesis and hematopoiesis. In the adult, the chemokine family of secreted proteins signals migration of hematopoietic cells through G-coupled chemokine receptors. We detected embryonic expression of chemokine receptor messages by RT-PCR with degenerate primers at embryonic day 7.5 (E7.5) or by RNase protection analyses of E8.5 and E12.5 tissues. In all samples, the message encoding CXCR4 was the predominate chemokine receptor detected, particularly at earlier times (E7.5 and E8.5). Other chemokine receptor messages (CCR1, CCR4, CCR5, CCR2, and CXCR2) were found in E12.5 tissues concordant temporally and spatially with definitive (adult-like) hematopoiesis. Expression of CXCR4 was compared with that of its only known ligand, stromal cell-derived factor-1 (SDF-1), by in situ hybridization. During organogenesis, these genes have dynamic and complementary expression patterns particularly in the developing neuronal, cardiac, vascular, hematopoietic, and craniofacial systems. Defects in the first four of these systems have been reported in CXCR4- and SDF-1-deficient mice. Our studies suggest new potential mechanisms for some of these defects as well as additional roles beyond the scope of the reported abnormalities. Earlier in development, expression of these genes correlates with migration during gastrulation. Migrating cells (mesoderm and definitive endoderm) contain CXCR4 message while embryonic ectoderm cells express SDF-1. Functional SDF-1 signaling in midgastrula cells as well as E12.5 hematopoietic progenitors was demonstrated by migration assays. Migration occurred with an optimum dose similar to that found for adult hematopoietic cells and was dependent on the presence of SDF-1 in a gradient. This work suggests roles for chemokine signaling in multiple embryogenic events.

Interleukin 3 enhances cytotoxic T lymphocyte development and class I major histocompatibility complex "re-presentation" of exogenous antigen by tumor-infiltrating antigen-presenting cells.

We show that interleukin 3 (IL-3) enhances the generation of tumor-specific cytotoxic T lymphocytes (CTLs) through the stimulation of host antigen-presenting cells (APCs). The BALB/c (H-2d) spontaneous lung carcinoma line 1 was modified by gene transfection to express ovalbumin as a nominal "tumor antigen" and to secrete IL-3, a cytokine enhancing myeloid development. IL-3-transfected tumor cells are less tumorigenic than the parental cell line, and tumor-infiltrating lymphocytes isolated from these tumors contain increased numbers of tumor-specific CTLs. By using B3Z86/90.14 (B3Z), a unique T-cell hybridoma system restricted to ovalbumin/H-2b and implanting the tumors in (BALB/c x C57BL/6)F1 (H-2d/b) mice, we demonstrate that the IL-3-transfected tumors contain an increased number of a rare population of host cells that can process and "re-present" tumor antigen to CTLs. Electron microscopy allowed direct visualization of these host APCs, and these studies, along with surface marker phenotyping, indicate that these APCs are macrophage-like. The identification of these cells and their enhancement by IL-3 offers a new opportunity for tumor immunotherapy.