Transforming growth factor ␤ (TGF-␤) is implicated in the regulation of smooth muscle cell (SMC) differentiation. We previously identified a novel TGF-␤ control element (TCE) in the promoters of SMC differentiation marker genes, including ␣-smooth muscle actin and SM22␣. In this study, the importance of the TCE in regulation of SM22␣ gene expression in vivo was investigated by mutating it within the context of a mouse SM22␣ promoter-lacZ transgenic construct. Mutation of the TCE completely abolished SM22␣ promoter activity in arterial SMCs as well as in developing heart and skeletal muscle. To identify the transcription factor(s) binding to the TCE, we performed yeast one-hybrid cloning analysis and identified gut-enriched Krü ppellike factor (GKLF). However, cotransfection studies in cultured cells showed that GKLF repressed the TGF-␤-dependent increases in SM22␣ and ␣-smooth muscle actin promoter activities. Furthermore, GKLF was not highly expressed in differentiated SMCs in vivo, and TGF-␤ down-regulated GKLF expression in dedifferentiated cultured SMCs. In contrast, overexpression of a related factor (BTEB2) transactivated SM22␣ promoter activity. Thus, our findings suggest a reciprocal role for related Krü ppel-like transcription factors in the regulation of SMC differentiation through a TCE-dependent mechanism.

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OX3 9DS, UKhAG-2 and hAG-3 are recently discovered human homologues of the secreted Xenopus laevis proteins XAG-1/2 (AGR-1/2) that are expressed in the cement gland, an ectodermal organ in the head associated with anteroposterior fate determination during early development. Although the roles of hAG-2 and hAG-3 in mammalian cells are unknown, both proteins share a high degree of protein sequence homology and lie adjacent to one another on chromosome 7p21. hAG-2 mRNA expression has previously been demonstrated in oestrogen receptor (ER)-positive cell lines. In this study, we have used real-time quantitative RT -PCR analysis and immunohistochemistry on tissue microarrays to demonstrate concordant expression of hAG-2 and hAG-3 mRNA and protein in breast tumour tissues. Tumour expression of both genes correlated with OR (hAG2, P ¼ 0.0002; hAG-3, P ¼ 0.0012), and inversely correlated with epidermal growth factor receptor (EGFR) (P ¼ 0.003). Yeast two-hybrid cloning identified metastasis-associated GPIanchored C4.4a protein and extracellular alpha-dystroglycan (DAG-1) as binding partners for both hAG-2 and hAG-3, which if replicated in clinical oncology would demonstrate a potential role in tumour metastasis through the regulation of receptor adhesion and functioning. hAG-2 and hAG-3 may therefore serve as useful molecular markers and/or potential therapeutic targets for hormone-responsive breast tumours.

Proteins associated with cancer cell plasma membranes are rich in known drug and antibody targets as well as other proteins known to play key roles in the abnormal signal transduction processes required for carcinogenesis. We describe here a proteomics process that comprehensively annotates the protein content of breast tumor cell membranes and defines the clinical relevance of such proteins. Tumor-derived cell lines were used to ensure an enrichment for cancer cell-specific plasma membrane proteins because it is difficult to purify cancer cells and then obtain good membrane preparations from clinical material. Multiple cell lines with different molecular pathologies were used to represent the clinical heterogeneity of breast cancer. Peptide tandem mass spectra were searched against a comprehensive data base containing known and conceptual proteins derived from many public data bases including the draft human genome sequences. This plasma membrane-enriched proteome analysis created a data base of more than 500 breast cancer cell line proteins, 27% of which were of unknown function. The value of our approach is demonstrated by further detailed analyses of three previously uncharacterized proteins whose clinical relevance has been defined by their unique cancer expression profiles and the identification of proteinbinding partners that elucidate potential functionality in cancer.

Abstract-Transforming growth factor-␤ (TGF-␤) has been shown to stimulate smooth muscle (SM) ␣-actin expression in smooth muscle cells (SMCs) and non-SMCs. We previously demonstrated that the 2 CArG boxes A and B and a novel TGF-␤ control element (TCE) located within the first 125 bp of the SM ␣-actin promoter were required for TGF-␤ inducibility of SM ␣-actin in SMCs. The aims of the present study were (1) to determine whether the TCE exhibits SMC specificity or contributes to TGF-␤ induction of SM ␣-actin expression in non-SMCs (ie, endothelial cells and fibroblasts) and (2) to determine whether TGF-␤ can induce expression of multiple TCE-containing SMC differentiation marker genes, such as SM22␣, h 1 calponin, and SM myosin heavy chain (SM MHC) in non-SMCs. Results of transient transfection assays demonstrated that mutation of CArG A, CArG B, or the TCE within a 125-bp promoter context completely abolished TGF-␤ inducibility of SM ␣-actin in endothelial cells and fibroblasts. However, in contrast to observations in SMCs, inclusion of regions upstream from Ϫ155 completely repressed TGF-␤ responsiveness in non-SMCs. Electrophoretic mobility shift assays showed that TGF-␤ enhanced binding of a serum response factor to the CArG elements and the binding of an as-yet-unidentified factor to the TCE in endothelial cells and fibroblasts, but to a much lesser extent compared with SMCs. TGF-␤ also stimulated expression of the SMC differentiation marker SM22␣ in non-SMCs. However, in contrast to SMCs, TGF-␤ did not induce expression of h 1 calponin and SM MHC in non-SMCs. In summary, these results suggest a conserved role for CArG A, CArG B, and the TCE in TGF-␤-induced expression of SM ␣-actin in SMCs and non-SMCs that is modified by a complex interplay of positive-and negative-acting cis elements in a cell-specific manner. Furthermore, observations that TGF-␤ stimulated expression of several early but not late differentiation markers in non-SMCs indicate that TGF-␤ alone is not sufficient to induce transdifferentiation of non-SMCs into SMCs.