Functional analysis of the human Sprouty2 gene promoter☆
Introduction
The mammalian lung and the Drosophila respiratory tracheal system both develop by branching morphogenesis, a well-patterned process which depends on epithelial–mesenchymal interactions mediated by several signaling pathways (Warburton et al., 2000). Members of the fibroblast growth factor (FGF) family, especially FGF10, are one of the inducing agents involved in this phase of embryonic development, since FGF10-deficient mice exhibit complete absence of branched lungs with just a blind-ended trachea remaining Sekine et al., 1999, Bellusci et al., 1997. Sprouty was identified as an antagonist of FGF signaling Sutherland et al., 1996, Hacohen et al., 1998.
Sprouty was originally discovered in Drosophila using genetic screens (Hacohen et al., 1998). Loss of function mutations of dSprouty led to overactive FGF signaling and excessive tracheal branching, whereas engineered overexpression of dSprouty during the primary branch stage blocked all secondary branching (Hacohen et al., 1998). Subsequent studies have shown that dSprouty inhibits signaling mediated not only by the FGF receptor (FGFR), but also several other receptor tyrosine kinases (RTK), such as epidermal growth factor receptor (EGFR), Sevenless and Torso Casci et al., 1999, Kramer et al., 1999, Reich et al., 1999. Although dSprouty has been suggested to act as an inhibitor of the RTK-induced mitogen-activated protein kinase (MAPK) signaling pathway, the molecular mechanism underlying this inhibitory activity remains unknown.
While Drosophila has only one Sprouty gene, at least four Sprouty homologs (Spry1-4) have been cloned in human as well as mouse Hacohen et al., 1998, de Maximy et al., 1999, Minowada et al., 1999, Chambers and Mason, 2000. All Sprouty proteins share a highly conserved, cysteine-rich C-terminal domain and a more divergent N-terminus. Among the four isoforms, the biological role of Spry2 has been characterized as a negative regulator of the FGF signaling pathways in modeling different varieties of branching tissues during development (Mailleux et al., 2001). In chick and mouse, overexpression of Spry2 leads to a reduction in limb development and bone outgrowth, phenotypes that resemble those induced by FGFR3 mutations (Minowada et al., 1999). Moreover, constitutive expression of Spry2 in the peripheral lung epithelium of transgenic mice results in decreased branching and inhibition of epithelial proliferation (Mailleux et al., 2001). Conversely, abrogation of Spry2 expression with antisense oligonucleotides stimulates branching in embryonic lung cultures (Tefft et al., 1999). In vitro, Spry2 inhibits FGF-induced differentiation of HUVEC and PC12 cells as well as migration of HeLa cells Impagnatiello et al., 2001, Gross et al., 2001, Yigzaw et al., 2001. However, it is interesting that the inhibitory effect of Drosophila Sprouty on EGF-induced signaling appears not to be iterated in mammalian systems. Instead, Spry2 inhibits EGFR endocytosis and subsequently potentiates the EGF-induced activation of MAP kinase by direct interacting with c-Cbl Wong et al., 2002, Sasaki et al., 2001.
By Northern analysis, the murine Spry2 mRNA is most abundant in lung, brain and heart followed by kidney and skeletal muscle (Tefft et al., 1999). Interestingly, a close correlation of Spry2 expression with known sites of FGF activity was found in the developing chick embryo (Chambers and Mason, 2000). Recent studies further demonstrated that Spry2 is expressed in highly restricted and dynamic temporo-spatial patterns during mouse embryonic lung development (Mailleux et al., 2001). Spatially, the expression of Spry2 is localized predominantly at the distal epithelium. Temporally, the mRNA level of Spry2 increases as the lung bud grows, peaking just before bud growth arrests and then sharply drops in the cleft when the mother bud splits laterally to give rise to two daughter buds. Both in time and space, the expression domains of Spry2 are adjacent to that of Fgf10 in the peripheral mesenchyme (Mailleux et al., 2001). These results suggest that transcriptional regulation of Spry2 expression is important for maintenance of the balance between the stimulatory factor FGF10 and inhibitory factor Spry2 in the developing lung. The purpose of this study is to determine and characterize the promoter of the human Spry2 gene and, hence, further elucidate the molecular mechanisms underlying the regulation of hSpry2 expression.
Section snippets
Cell culture
Human A549 (lung carcinoma) epithelial cells, human fetal lung WI-38 VA-13 fibroblasts (SV40 virus-transformed derivative of WI-38) and human HeLa (cervix carcinoma) cells were cultured in Dulbecco's modified Eagles' medium (DMEM) supplemented with 10% fetal calf serum (FCS). WI-38 VA-13 cells were obtained from American Type Culture Collection (Manassas, VA).
Rapid amplification of cDNA ends
Transcription start sites were identified by 5′-RACE, following the protocol from the GeneRacer kit (Invitrogen). The human fetal lung
Identification of the transcription start sites of hSpry2
We used 5′-RACE to localize the position of the hSpry2 transcription start site(s). 5′-RACE was performed using poly(A)+ RNA from fetal human lungs and a hSpry2-specific primer. A strong single band of about 500 bp was obtained after amplification. This 5′-RACE product was subsequently subcloned and sequenced. From a total of 24 isolates, 23 clones contained sequences homologous to hSpry2. Two end points that are separated by six nucleotides were detected, suggesting the existence of multiple
Discussion
This is the first study of any mammalian Sprouty promoters, therefore no comparison of functional motifs conserved between species or between Sprouty isoforms can be made. Nonetheless, in the present work, we characterized the 5′-flanking region of the human Spry2 gene, which is responsible for its transcriptional regulation in cell culture. We have focused mainly on the identification of the promoter elements involved in constitutive gene expression. Using luciferase reporter gene assays from
Supplementary Files
Acknowledgements
This work was supported in part by research training fellowship from the American Lung Association (W.D.) and in part by HL 44060, HL 44977 and HL 60231 (D.W).
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2009, BloodCitation Excerpt :SPRY2 expression was induced in a dose-dependent manner in 2F7, P3HR1, and BL41 BL cells with 5-aza-dC with or without TSA treatment (Figure 4C), further supporting a role for epigenetic control of SPRY2 expression. A direct role for dense DNA hypermethylation in repressing SPRY2 expression was examined using increasing-length SPRY2 promoter-luciferase reporter constructs41 that were unmethylated or hypermethylated in vitro at CG dinucleotides with SssI methyltransferase and transfected into human embryonic kidney (HEK) 293T cells (Figure S2A). Dense CpG methylation of all 3 reporter constructs resulted in almost complete suppression of SPRY2 promoter activity, with luciferase activity reduced 50- to 200-fold compared with unmethylated reporter constructs (Figure S2B).
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Supplementary data associated with this article can be found at doi: 10.1016/j.gene.2003.09.004.