Platelet-derived growth factors (PDGFs) 1 are major mitogens for connective tissue cells that are involved in diverse biological processes including physiological development, tissue repair, tumorigenesis, and atherosclerosis (1). PDGF family members, PDGF-A, -B, -C, and -D, are assembled as disulfide-linked homo-or heterodimers and exert their activity by binding to and activating specific high affinity cell surface receptors. Two receptor subtypes with protein-tyrosine kinase activity have been identified that can form homo-and heterodimeric receptor complexes: the ␣-subunit, which can bind to the A-, B-, and C-chains of PDGF, and the ␤-subunit, specific for the B-, C-and D-chains (1-4). Dermal fibroblasts are one of the major target cells of PDGF in the initiation and propagation of wound healing in the skin (5). Levels of PDGF receptor (PDGFR)-␣ expression are high in fibroblasts during early embryogenesis, and disruption of PDGFR-␣ results in a reduction in fibroblasts throughout the embryo (6, 7). In contrast, targeted deletion of PDGFR-␤ and analysis of blastocyst chimeras demonstrated no effect of PDGFR-␤ on fibroblast development (8, 9). However, in mice prepared from the blastocyst chimeras that contain a combination of wild-type and PDGFR-␤ Ϫ/Ϫ cells, analysis of granulation tissue formation following the subcutaneous implantation of sponges demonstrated that PDGFR-␤ Ϫ/Ϫ cells were depleted in the granulation tissue (10). These reports suggest that the two subtypes of PDGFR play distinct roles in development and that PDGFR-␤ is important in wound healing by dermal fibroblasts. Analysis of mutant mice in which the cytoplasmic signaling domain of the PDGFR-␤ was used to replace the PDGFR-␣ cytoplasmic domain showed no obvious defects in any of the PDGFR-␣-dependent cell types (11). On the other hand, when the PDGFR-␤ was dependent upon PDGFR-␣ cytoplasmic domain, multiple abnormalities occurred in vascular smooth muscle cell development (11). These data suggest that PDGFR-␤ has unique signaling capacities compared with PDGFR-␣.PDGF binding to the receptors activates a variety of intracellular signaling molecules (12). One of these is phosphatidylinositol 3-kinase (PI3K), which results in the local accumulation of PI(3,4,5)P 3 at the plasma membrane. Synthesized * This study was supported in part by Grants-in-aid for Scientific Research 16390114 and 12470053 from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ § To whom correspondence should be addressed: Dept.

For identification of the antigens specific to Marek's disease virus (MDV) in virus-non-producer lymphoblastoid cell lines established from a tumor of Marek's disease (MD), hybridomas producing monoclonal antibodies (MAbs) against the antigens were isolated. Immunogens for preparation of the hybridomas were purified from the lysate of an MD-lymphoblastoid cell line, MSB1, by affinity chromatography coupled with chicken anti-MDV serum immunoglobulin G. Three of the MAbs obtained, MB1, MB2 and MB3, were specific to MDV by immunofluorescence test. An immunofluorescence test using MB2 antibody showed that immunofluorescence-positive cells in non-producer MD-lymphoblastoid line cells became detectable when the culture temperature was shifted from 41 degrees C to 33 degrees C or when treatment with 5-iodo-2-deoxyuridine (IUdR) was performed, indicating that the antigen reactive with MB2 antibody is an MDV-specific early antigen. This temperature shift or IUdR-treatment did not induce other MDV-specific antigens, such as late gene products of MDV, detected with MAbs. MB1 and MB2 antibodies immunoprecipitated 4 MDV-specific phosphorylated polypeptides with molecular weights (MWs) of 43,000 (43kd), 39kd, 36kd and 24kd from chick embryo fibroblasts productively infected with virulent MDV. In the place of 43kd, phosphorylated 44kd polypeptide was precipitated from avirulent MDV-infected fibroblasts. However, MB3 antibody did not precipitate any MDV-specific polypeptides from infected fibroblasts. These results suggest that the phosphorylated polypeptides are MDV-specific polypeptides predominantly expressed in non-producer MD-lymphoblastoid cell lines.

SUMMARYBy use of monoclonal antibodies, a virus-specific cytoplasmic antigen related to phosphorylated polypeptides specific to serotype 1 of Marek's disease virus (MDV)-related viruses (MDV1) has been identified in all MD tumour cell lines examined, as well as in infected cells and in tumour lesions of chickens with MD. At least two phosphorylated polypeptides with mol. wt. 39000 (39K) to 36K and 24K (pp39/36 and pp24, respectively) were identified in the MD tumour cell line H10 cultured at 33 °C by immunoprecipitation with monoclonal antibody M21 which reacts with virus-specific phosphorylated polypeptides. These polypeptides were not detected in cells infected with MDV-related viruses of serotype 2 or 3. Immunoblot analysis indicated that these two polypeptides contained a serotype 1-specific epitope recognized with M21. An additional 41K polypeptide appeared in different virus strains of serotype 1. These polypeptides were found to contain phosphorylated serine but no detectable phosphorylated tyrosine or phosphorylated threonine. Cell fractionation indicated that the two phosphorylated polypeptides were mainly associated with smooth and rough endoplasmic reticulum fractions of cells infected with MDV1. Furthermore, the mRNA coding for pp39/36 could be separated from that coding for pp24 on a sucrose density gradient. These results suggest that pp24 and pp39/36 are translated from distinct mRNAs and encoded from overlapping genes or separate regions with partial DNA homology in the MDVI genome.

Insulin resistance and smoking are significant risk factors for cardiac and cerebral vascular diseases. Because vascular smooth muscle cells play a key role in the development and progression of atherosclerosis, we investigated the effect of nicotine on insulin-induced mitogenic signaling in aortic vascular smooth muscle cells isolated from Sprague Dawley rats. RT-PCR revealed the expression of alpha2-7, alpha10, beta1-3, delta, and epsilon subunits of the nicotinic acetylcholine receptor (nAChR) in the cells. Short-term nicotine treatment stimulated phosphorylation of p44/42-MAPK, p38-MAPK, and signal transducer and activator of transcription 3. However, an additive effect of nicotine pretreatment on insulin stimulation was only observed on p44/42-MAPK. The nicotine-induced phosphorylation of p44/42-MAPK and [methyl-(3)H]thymidine incorporation were effectively suppressed by a alpha7-nAChR-selective antagonist, methyllycaconitine, and the phosphorylation of p44/42-MAPK was stimulated by a alpha7-nAChR-specific agonist, GTS21. Furthermore, the phosphorylation was mediated via calmodulin kinase II, Src, and Shc. Interestingly, long-term (48-h) pretreatment with nicotine increased the amount of alpha7-AChR in the plasma membrane and insulin-induced phosphorylation of p44/42-MAPK. These results provide the first evidence that acute exposure to nicotine enhances insulin-induced mitogenesis predominantly by affecting the phosphorylation of p44/42-MAPK and that chronic exposure further augments the insulin signal via up-regulation of alpha7-nAChR, which may be crucial for the development and progression of atherosclerosis in large vessels.

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