Using a reporter gene (secreted alkaline phosphatase, SPAP) immediately downstream of a synthetic promoter containing 6 cyclic AMP response element (CRE) sequences we have previously shown that a range of ß-adrenoceptor (AR) agonists stimulate CRE-mediated gene transcription via the human ß2-adrenoceptor in CHO-K1 cells (Baker et al., 2003a). Here, we investigate the temporal characteristics of isoprenaline-induced (Serine 133) phosphorylation of CRE binding protein (CREB), to determine whether isoprenaline can induce gene transcription via a natural promoter and finally whether endogenous protein expression can be detected.

CHO-K1 cells expressing the human ß2-AR were secondarily transfected with a pGL3 reporter vector containing the full c-fos promoter (which contains a single CRE; Hill et al., 2001) upstream of a luciferase reporter gene as described previously (CHO-ß2-fos; Megson et al., 2001). Cells were stimulated with agonist (5hr) and luciferase activity measured as previously described (Baker et al., 2003b). CHO-ß2 cells (Baker et al., 2002) were stimulated for 5min-5hr with agonist before being solubilized and whole cell extract used for Western blotting. Blots were incubated with primary antibody (anti-phospho-CREB (ser-133); anti-CREB; the pan anti-c-fos-K25 (c-fos and Fra-1); the selective anti-c-fos4 (for c-fos only) and the selective anti-Fra-1) for 2hrs (room temperature) in 5% BSA. HRP-conjugated donkey anti-rabbit secondary antibody was incubated for 1hr before immuno-reactivity detected with enhanced chemiluminescence.

In CHO-ß2-fos cells, isoprenaline stimulated an increase in c-fos-luciferase expression (log EC₅₀ = -8.25 + 0.16, n=8). orskolin also stimulated an increase in c-fos-luciferase expression (log EC₅₀ = -6.13 + 0.15, n=4) thus demonstrating that increasing cAMP either directly or via the ß2-AR can stimulate gene expression via a natural promotor.

Isoprenaline (10µM) induced a rapid (10mins) and sustained increase in phospho-CREB accumulation which was maintained for 5 hrs (n=8) as seen on Western blotting. However, isoprenaline (10µM) produced a transient increase in the expression of c-fos protein (immediate early gene) between 30min to 2hrs of stimulation. This was detected with both c-fos antibodies (c-fos K25 n=7; and c-fos-4, n=4) thus demonstrating an increase in gene transcription of an endogenous protein. This is similar to the c-fos protein expression in CHO cells in response to insulin (Griffiths et al., 1998). Isoprenaline also produced a marked stimulation of Fra-1 expression which was detectable by both the pan c-fos-K25 and the Fra-1 antibody at 1hr following agonist stimulation and maintained up to 5hrs (c-fos K25 n=6; Fra-1 n=3). This was seen as an increase in intensity of a triplet of bands of apparent molecular mass 35-40 kDa (detectable with the K25 antibody) which has been shown previously to represent Fra-1 in CHO cells (Griffiths et al., 1998) and again demonstrates the production of an endogenous protein.

In summary, isoprenaline can stimulate gene transcription of endogenous proteins in CHO-ß2 cells.

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