Cycloheximide treatment to identify components of the transitional transcriptome in PACAP-induced PC12 cell differentiation

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neurite outgrowth, reduces proliferation and inhibits apoptosis of PC12 cells. We have partially characterized the transcriptome changes induced by PACAP after 6 h of treatment, when commitment to differentiation has occurred. Here, we have investigated the effects of a 6-h treatment with PACAP (10(-7) m) in the presence of cycloheximide (5 microm) to identify, via superinduction, components of the transitional transcriptome initially induced by PACAP and potentially participating in the regulation of late-response genes required for differentiation. Approximately 100 new transcripts were identified in this screen, i.e. as many individual genes as make up the 6-h PACAP differentiation transcriptome itself. Six known transcripts in this cohort were then measured at several time points between 0 and 6 h by real-time PCR to determine whether these transcripts are induced early following PACAP treatment in the absence of cycloheximide, and therefore may be of functional importance in differentiation. Five out of the six transcripts were indeed induced by PACAP alone soon (between 30 min and 3 h) after cell treatment. beta-Cell translocation gene 2, antiproliferative (Btg2), serum/glucocorticoid-regulated kinase (Sgk), nuclear factor for the kappa chain of B-cells (NFkappaB), seven in absentia homologue 2 (Siah2) and FBJ osteosarcoma related oncogene (Fos) showed a 2.5-200-fold induction by PACAP between 15 min and 3 h, and mRNA levels returned either to baseline or near baseline after 6 h. This work provides new information concerning genes whose transient regulation early after PACAP exposure may contribute to the expression of the differentiated transcriptome in PC12 cells, and should help to elucidate the molecular mechanisms involved in the control of nerve cell survival and differentiation.

Fig. 1

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) receptors mRNA in PC12 cells. (a) Expression of PAC1, VPAC1, VPAC2 and GAPDH receptor isoforms in either the absence or the presence of PACAP and/or cycloheximide. cDNA from hypothalamus and lung tissues were used as positive controls for PAC1 and VPAC receptors, respectively. (b) Quantification by real-time PCR of the effect of cycloheximide on PAC1 receptor expression after 30 min of treatment with cycloheximide.

Fig. 2

Functional classification according to the GeneOntology (GO) reference of the mRNA activated by pituitary adenylate cyclase-activating polypeptide (PACAP) plus cycloheximide (biological process level three). Cluster analysis was conducted with the FatiGO application (http://www.fatigo.org). As illustrated with FBJ osteosarcoma-related oncogene (Fos), which belongs to six categories indexed in GO biological process level three (pattern specification, sex differentiation, embryonic development and organ development; metabolism; morphogenesis, cell differentiation and cell development; regulation of physiological process, organismal physiological process; regulation of cellular process and finally cellular physiological process), one particular gene can be assigned to several GO annotations.

Fig. 3

Independent verification of representative genes regulated by pituitary adenylate cyclase-activating polypeptide (PACAP) in PC12 cells. Quantification of FBJ osteosarcoma-related oncogene (Fos), β-cell translocation gene 2, antiproliferative (Btg2), seven in absentia 2 (Siah2), serum/glucocorticoid-regulated kinase (Sgk), ubiquitin-conjugating enzyme E2D 3 (UBC4/5 homologue, yeast) (NFκB) and activity-dependent neuroprotective protein (Adnp) expression levels by real-time PCR after 6 h of treatment with either PACAP (10⁻⁷ m) alone or in the presence of cycloheximide (5 μm). Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA was used as an internal standard. ***p < 0.001; ns, not statistically different.

Fig. 4

Time-course of pituitary adenylate cyclase-activating polypeptide (PACAP) effect on gene expression. PC12 cells were exposed to PACAP (10⁻⁷ m) for durations ranging from 2 min to 48 h and the gene-expression profile was measured by real-time PCR for FBJ osteosarcoma-related oncogene (Fos), β-cell translocation gene 2, antiproliferative (Btg2), seven in absentia 2 (Siah2), serum/glucocorticoid-regulated kinase (Sgk), nuclear factor for kappa chain of B-cells (NFκB) and activity-dependent neuroprotective protein (Adnp). Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA was used as an internal standard. Solid lines correspond to the mRNA expression in PACAP-treated cells and dashed lines correspond to the mRNA expression in cells treated with control medium. **p < 0.01; ***p < 0.001; unmarked data points, not statistically different from control.