Analysis of the PC12 cell transcriptome after differentiation with pituitary adenylate cyclase‐activating polypeptide (PACAP)

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neurite outgrowth and inhibits proliferation of rat pheochromocytoma (PC12) cells. Characterizing the PACAP-differentiated PC12 cell transcriptome should provide genetic insight into how these processes occur in these cells, and in neuronal precursors in vivo. For this purpose, RNA samples were collected from PC12 cells before or after a 6-h treatment with PACAP, from which a labeled cDNA was hybridized to a high-density cDNA array containing 15 365 genes. The genomic response to PACAP involves at least 73 genes. Among the genes differentially expressed in the presence of PACAP, 71% were up regulated, and 29% down regulated, 2-fold or more. Sixty-six percent of the messages affected by PACAP code for functionally categorized proteins, most not previously known to be regulated during PC12 cell differentiation. PACAP has been shown to induce PC12 cell neurite outgrowth through the mitogen-activated protein kinase kinase (MEK) pathway independently of protein kinase A (PKA). Therefore treatments were conducted in the absence or presence of the PKA inhibitor H89, or the MEK inhibitor U0126 in order to identify subsets of genes involved in specific aspects of PC12 cell differentiation. Co-treatment of PC12 cells with PACAP plus H89 revealed a cluster of five genes specifically regulated through the PKA pathway and co-treatment of the cells with PACAP and U0126 revealed a cluster of 13 messages specifically activated through the MEK pathway. Many of the known genes regulated by PACAP have been associated with neuritogenesis (i.e. villin 2 or annexin A2) or cell growth (i.e. growth arrest specific 1 or cyclin B2). Thus, some of the expressed sequence tags (ESTs) that exhibit the same regulation pattern (i.e. AU016391 or AW552690) may also be involved in the neuritogenic and anti-mitogenic effects of PACAP in PC12 cells. Among the 73 PACAP regulated genes, 10 are disqualified on pharmacological grounds as actors in PACAP-mediated neurite outgrowth or growth arrest, leaving 63 new PACAP-regulated genes implicated in neuronal differentiation. Thirteen of these are candidates for mediating ERKdependent neurite outgrowth, and 47 are possibly involved in the ERK-independent growth arrest induced by PACAP.

Latent process genes for cell differentiation are common decoders of neurite extension length

“…Gene expression accompanied by cell differentiation induced by NGF, PACAP, forskolin and dibutyryl cAMP has been studied intensively (Dijkmans et al, 2008;Ng et al, 2009;Ravni et al, 2008;Vaudry et al, 2002a), and the neurotrophic factorresponsive genes that were highlighted potentially involve LP genes. LP genes might be some of the many genes whose expression is induced by neurotrophic factors and correlated with neurite extension length.…”

Pituitary adenylate cyclase-activating polypeptide prevents the effects of ceramides on migration, neurite outgrowth, and cytoskeleton remodeling

“…Previous studies have shown that PACAP-induced differentiation is mediated through cAMP production in all cell types investigated, including cerebellar granule cells (19), pheochromocytoma cells (29), neuroblastoma cells (30), and embryonic stem cells (31). Increased formation of cAMP induced by PACAP can likely account for its inhibitory effect on cell migration because somatostatin, a peptide that inhibits adenylyl cyclase activity, has been shown to stimulate the early phase of granule cell migration (22).…”

Regulation of PC12 Cell Differentiation by cAMP Signaling to ERK Independent of PKA: Do All the Connections Add Up?

“…Although it is tempting to propose this cAMP-EPAC-Rit signaling cassette as the noncanonical pathway mediating neuritogenesis, there still remains a crucial unconnected component: ERK. PACAP activation of ERK is well documented as essential for PACAP-mediated neuritogenesis in PC12 cells (17,19). However, knockdown of Rit did not prevent PACAP activation of ERK in PC6 cells (24), yet it did diminish (though not abolish) PACAP-mediated neurite formation.…”

“…PC12 cells adopt a neuronal-like morphology when treated with nerve growth factor (NGF) or with cAMP-elevating agents, such as forskolin or PACAP (15)(16)(17). The extension of neurites is one hallmark of the neuronal phenotype, along with cessation of proliferation; expression of differentiationspecific genes, such as those encoding transin and sodium-channel proteins; and production of specific neurotransmitters (18).…”

“…The extension of neurites is one hallmark of the neuronal phenotype, along with cessation of proliferation; expression of differentiationspecific genes, such as those encoding transin and sodium-channel proteins; and production of specific neurotransmitters (18). Based on pharmacological analysis, PACAP-mediated neuritogenesis is independent of PKA (not blocked by H89, a specific PKA inhibitor) but is dependent on ERK (blocked by UO126, a specific inhibitor of MEK, the kinase upstream of ERK that is required for ERK activation) (17,19) (Fig. 1).…”