We hypothesized that fibroblasts and possibly other abundant tissue cell types are major sources of sST2 protein in vivo and that deletion of the proximal promoter would result in less circulating sST2 and thus disruption of normal IL-33 regulation. Instead, we found that although loss of the proximal promoter abolished fibroblast-specific ST2 expression, it had no obvious impact on the amount of circulating sST2. Figure 1A is a map of the mouse ST2 locus illustrating the location of the two promoters, the intron-exon organization and the targeting strategy to generate the proximal promoter

and enhancer knockout. Figure 1B illustrates the alternative splicing whereby exons 9–11 are SB203580 supplier either included in the final spliced LDE225 order ST2L mRNA, or not included thereby leading to incorporation of an alternative stop codon and the generation of sST2. We selectively deleted the ST2 proximal promoter (with noncoding exon 1b) and its associated enhancer element. The resulting locus contains in their place a single loxP site, yet still retains the distal promoter and all coding exons. Homozygous knockout mice bred normally and nearly all animals lacked overt developmental or pathological

manifestations. However, interestingly, two homozygous knockout mice spontaneously developed what appeared to be subcutaneous tumors on Bay 11-7085 their neck and trunk and a third animal was found moribund due to unknown causes (not shown). Possibly relevant to these observations are previous findings that sST2 is correlated with progression of breast cancer [15] and that sST2 may modulate tumor cell activity in vitro [16]. Based on previous findings, we predicted that proximal promoter deletion would not disrupt expression of ST2L in immune cells. We performed a PCR designed to specifically amplify sST2 or

ST2L cDNAs, as indicated in Fig. 1A, and found that as expected ST2L mRNA was expressed similarly in both wild type and knockout splenocytes (Fig. 1C). Little to no expression of sST2 was detected in splenocytes. Therefore, consistent with previous data, we found splenocytes express predominantly the ST2L isoform and deletion of the proximal promoter did not abolish ST2L expression. We also found that deletion of the proximal promoter had minimal effects on the expression of ST2 in bone marrow-derived mast cells (BMMCs) (Fig. 1C). BMMCs express both sST2 and ST2L transcripts and neither isoform was affected by promoter deletion. Also, BMMCs from knockout mice developed normally in vitro (based on c-kit expression) and expressed equivalent amounts of ST2L on the cell surface compared with wild-type BMMCs (Fig. 1D). Moreover, knockout BMMCs responded to IL-33 by secreting equivalent amounts of IL-6 as compared with wild-type BMMCs (Fig. 1E).