Identification of a Series of Transforming Growth Factor β-Responsive Genes by Retrovirus-Mediated Gene Trap Screening

Abstract

Transforming growth factor ␤ (TGF-␤) plays important roles in the regulation of proliferation, differentiation, apoptosis, and carcinogenesis. To identify genes responsible for maintaining the phenotype induced by TGF-␤, we performed a retrovirus-mediated gene trap screening designed to isolate TGF-␤-responsive genes in human lung carcinoma cell line A549. After screening 249 trap lines, 21 were found to express the reporter ␤-galactosidase gene in a TGF-␤-responsive manner. Interestingly, in large proportions of these trap lines, the reporter gene was responsive also to phorbol ester and was suppressed by gamma interferon. Fragments of all these trapped genes were recovered by 5-and 3-rapid amplification of cDNA ends (RACE), and in 15 out of 21 cases (71%), the TGF-␤ responsiveness of the endogenous genes was confirmed by RNA blot hybridization. In at least five cases, the TGF-␤-induced upregulation was found to be cycloheximide resistant, suggesting the roles of the genes in the TGF-␤-induced primary responses. Sequence analyses revealed that 43% (9 of 21) of the trapped genes were novel and that the remainder included genes previously reported to be upregulated by TGF-␤, such as epidermal growth factor receptor and ␤1 integrin, documenting the validity of this approach. Other known genes include the ones encoding the proteins associated with cell proliferation (ribosomal proteins S15a, hNRP/NAP-1, and lipocortin II), focal adhesions (paxillin), and transcriptional regulation (thyroid hormone receptor activator molecule 1 [TRAM-1]).

Retracted: Ribosomal Protein S15A Augments Human Osteosarcoma Cell Proliferation In Vitro

“…[5][6][7] RPS15A has been shown to be upregulated in response to the Myc oncogenic transcription factor or transforming growth factor-b induction. 8,9 Recent studies have indicated that RPS15A is implicated in several human cancers. RPS15A is differentially expressed between poorly differentiated ductal carcinoma in situ and invasive grade 3 breast tumors, indicating that RPS15A may contribute to the progression of breast cancer.…”

Paxillin: Adapting to Change

“…Hic-5 also demonstrates widespread expression (247), although it is largely absent from leukocyte cell-rich tissues like spleen and thymus (328), whereas leupaxin is restricted to leukocyte populations (150). The genomic promoter elements of paxillin, Hic-5, and leupaxin have not been characterized; however, similar to Hic-5 (247), paxillin mRNA is induced by TGF-␤ as determined using a retrovirusmediated gene trap screen (2). Hic-5 mRNA expression is induced by retinoic acid, with Hic-5 then potentiating retinoic acid-dependent differentiation processes (249).…”

Gene trap screening as an effective approach for identification of Wnt‐responsive genes in the mouse embryo

“…In a modified genetrap strategy, called induction gene trap, genes are selected by their response to specific secreted signal proteins, including BMP2, activin, nodal, and FGF, added to the culture of genetrapped cells (Forrester et al, 1996;Harrison and Miller, 1996;Stoykova et al, 1998;Mainguy et al, 2000;Akiyama et al, 2000;Medico et al, 2001;Kluppel et al, 2002;Tateossian et al, 2004). Although the in vivo correlation between selected genes and signals has remained unclear in these cases, in vitro prescreening of trapped ES cell lines with secreted signal proteins would be effective for enrichment of genes regulated by specific signals even in normal development.…”