During differentiation of the nervous system, pluripotent neural stem cells give rise to a wide variety of neuronal and glial cell types. This differentiation involves the dynamic interplay of extrinsic environmental signals, cell-cell interactions, and intrinsic transcriptional regulatory events. The bone morphogenic proteins interact with complementary regional signals such as fibroblast growth factors, and sonic hedgehog to regulate early stages of neural stem cell expansion, self-renewal, lineage restriction, and incipient lineage commitment. The ability of these trophic signals to act within neurodevelopmental niches requires precise expression of members of the basic helix-loop-helix (bHLH) transcription factor family (reviewed in Takahashi and Liu 2006). bHLH factors regulate the fate of neural progenitor cells by controlling proliferation, cell cycle exit, neurite outgrowth, and synaptogenesis (Sun et al. 2001; Nguyen and Woo 2003). Gain- and loss-of-function studies have shown that precise temporal and spatial expression of bHLH transcription factors is critical for proper development of the nervous system (Casarosa et al. 1999).
As a bHLH factor, mammalian achaete-schute homolog 1 (Ascl1 or Mash1) is essential for the survival of neural progenitor cells, and plays a central role in generating neuronal diversity by regulating subtype specification as well as differentiation (Bertrand et al. 2002). Ascl1 is one of the earliest markers expressed in a subset of neural progenitor cells (Parras et al. 2004), and in the embryonic ventral telencephalon is essential for the production of neuronal precursor cells (Casarosa et al. 1999; Nieto et al. 2001). In the dorsal telencephalon, Ascl1, in concert with other proneural bHLH proteins from the Neurogenin family, promotes the neuronal commitment of multipotent progenitors while inhibiting their astrocytic differentiation (Nieto et al. 2001).
Previous research utilizing P19 embryonic carcinoma cells has shown that these cells function as pluripotent stem cells. Once induced to differentiate into neurons by retinoic acid and aggregation, they exhibit biochemical and developmental processes similar to those that occur in early embryogenesis. Furthermore, they share several properties in common with embryonic stem cells isolated from mice and humans (Thomson and Marshall 1998). Remarkably, transient transfection of NeuroD2, Ascl1, Neurog1 and related proneural bHLH proteins has shown that these key transcription factors are sufficient to convert uncommitted P19 cells into differentiated neurons (Farah et al. 2000). The consequences of Ascl1 expression in P19 cells are similar to those observed in vivo (Gowan et al. 2001): the differentiation of these transfected cells is preceded by elevated expression of the cyclin-dependent kinase inhibitor p27kip1 and cell cycle withdrawal. Furthermore, these differentiated neurons exhibit electrophysiological properties of neurons (Farah et al. 2000; Huang et al. 2010). However, little is known about the signaling cascades triggered downstream of Ascl1 that are involved in the differentiation and eventual function of these cells.
As a modulator of the sonic hedgehog and bone morphogenic protein pathways, cAMP-dependent protein kinase (PKA) is an essential integrator of signaling pathways (Tiecke et al. 2007; Ohta et al. 2008; Ghayor et al. 2009; Pan et al. 2009). During development, the cAMP/PKA pathway is critically involved in regulation of gene expression, cell growth, and cell differentiation. At low levels of cAMP, PKA exists as a tetrameric holoenzyme composed of two catalytic subunits and two regulatory subunits. Two genes encoding catalytic subunits of PKA have been identified in mammalians, designated Cα and Cβ (Lee et al. 1983; Uhler et al. 1986; Hedin et al. 1987). Four genes encoding the regulatory subunits of PKA are grouped into two categories: type I and type II. The type II regulatory subunits (RIIα and RIIβ) contain an autophosphorylation site (Hofmann et al. 1975; Rosen and Erlichman 1975), whereas the type I subunits (RIα and RIβ) are not autophosphorylated. The regulatory subunits are modular, highly dynamic proteins that bind to two molecules of cAMP, which results in their dissociation from the catalytic subunits of PKA. These free catalytic subunits then go on to phosphorylate specific serine or threonine residues on PKA substrates, eliciting changes in their biological function (Corbin et al. 1988; Taylor et al. 1990). In addition, the regulatory subunits also serve to specifically target the PKA holoenzyme to the A-kinase anchoring proteins within the cell (Banky et al. 1998; Newlon et al. 1999).
In addition to the regulatory and catalytic subunits, the protein kinase inhibitor (PKI) proteins are important physiological regulators of PKA (Dalton and Dewey 2006). Three genes encoding different isoforms of PKIs (PKIα, PKIβ and PKIγ) have been characterized in mammals and these genes show conserved tissue-specific expression (Collins and Uhler 1997; Zheng et al. 2000). PKIs were first identified as competitive inhibitors of the catalytic subunits and proposed to modulate the threshold for activation of PKA by cAMP (Ashby and Walsh 1972). Later, PKIs were also shown to cause translocation of the catalytic subunit from the nucleus to the cytoplasm (Wen et al. 1994). PKIγ has been shown to be required for the termination of immediate early gene induction by PKA (Chen et al. 2005) and PKIα has been shown to suppress the Nodal-Pitx2 pathway in chick embryos (Kawakami and Nakanishi 2001). In this study, we characterized PKA activation in P19 cells and demonstrated induction of all three isoforms of PKI during Ascl1-induced P19 neuronal differentiation. The magnitude of induction varied by isoform, and each PKI transcript also exhibited a distinct temporal pattern of expression. Short hairpin RNA (shRNA)-mediated knockdown of each isoform showed that PKIβ– the most highly induced isoform in our model system – and its inhibition of PKA activity is necessary for Ascl1-induced neuronal differentiation in P19 cells.
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cAMP-dependent protein kinase is critical for phenotypic specification and transition in the adult and developing nervous system, but its role in neuronal differentiation remains controversial with contradictory roles emerging depending on cell type. In vitro, PKA activity inhibits neuronal differentiation in SH-SY5Y human neuroblastoma cells by blocking the initial steps of neurite elongation (Canals et al. 2005), and in NG108-15 cells, PKA activity also appears to inhibit neuritogenesis and neurite outgrowth rate (Tojima et al. 2003). In vivo, PKA has been shown to effectively inhibit the progression of retinal neurogenesis in zebrafish via effects on cell cycle exit (Masai et al. 2005). Conflicting research shows that in SH-SY5Y cells, PKA activity is necessary for the initial steps of cAMP-induced neurite elongation (Sanchez et al. 2004). Similarly, in hippocampal HiB5 cells, treatment with a cAMP analog results in a dramatic increase in neurite outgrowth (Kim et al. 2002). The importance of downstream effects of PKA activity were highlighted in a study where a dominant-negative inhibitor of CREB was shown to be effective in attenuating nerve growth factor-mediated differentiation of PC12 cells (Ahn et al. 1998). Despite these incongruities, these data suggest that the level of active PKA expressed in a neuronal cell can have profound effects on neurite formation, which in turn can alter the excitability of a cell and its ability to generate and transfer electrical signals within the nervous system. Examination of the PKA-CREB signaling pathway in P19 cells showed that 8-CPT-cAMP was capable of activating the PKA pathway, as evidenced by increased levels of pCREB. Exogenous Cα also produced activation of a CRE-reporter (Fig. 1). During Ascl1-induced neuronal differentiation, a kinase assay demonstrated that P19 cells undergo a significant, but transient decrease in PKA activity early in the differentiation process (Fig. 2).
Measurement of basal kinase activity during Ascl1-induced neuronal differentiation demonstrated a significant reduction in basal kinase activity. As PKI proteins have the ability to inhibit basal kinase activity, we characterized the expression of these proteins. In our studies, following over-expression of Ascl1, microarray hybridization showed that P19 cells undergo a transient increase in all three isoforms of PKI, each displaying a unique temporal pattern of expression. We verified these results using qRT-PCR and found that the PKIβ transcript was the most highly induced, exhibiting a 2500-fold increase in expression (compared to an 18-fold and 6-fold expression for PKIα and PKIγ, respectively) that corresponded to the 2.7-fold decrease in PKA activity. We confirmed that the induction of PKIβ mRNA expression was accompanied by a significant increase in PKIβ protein (Fig. 4). As PKIs are specific inhibitory regulators of PKA, we hypothesized that PKIs could be responsible for the observed inhibition of PKA activity during Ascl1-mediated differentiation. shRNA constructs targeting each isoform were evaluated for their ability to knockdown expression of all three PKI genes, and although we successfully identified a number of effective shRNAs for each isoform, only those targeting the PKIβ gene prevented neuronal differentiation (compare Fig. 3 with Fig. 5). qRT-PCR analysis determined that the shRNA-mediated reduction in PKIβ mRNA and protein did not affect early events of Ascl1-mediated differentiation (e.g. Ascl1 and Gadd45γ induction), and also did not significantly affect the expression of other PKI isoforms (Fig. 6). The alternative splice variant of PKIβ induced in P19 cells is a specific inhibitor of PKA (Fig. 7) and the shRNA blockage of neuronal differentiation was partially rescued by over-expressing PKIβ protein. We found that this rescue of neuronal differentiation was dependent on four amino acid residues critical for binding of PKIβ to the catalytic subunit of PKA (Figs 7 and 8). As compensatory up-regulation of other PKI isoforms has been reported previously (Belyamani et al. 2001), we also conducted experiments testing whether exogenous PKIα or PKIγ expression could rescue the phenotype conferred by antisense knockdown of PKIβ. Our results suggest a requirement for PKIβ and its association with PKA during the neuronal differentiation of P19 cells. Importantly, the observation that neither PKIα nor PKIγ were able to rescue the block to neuronal differentiation caused by PKIβ shRNAs demonstrates a unique role for PKIβ (Fig. 9). Finally, very few culture systems have been described in which PKI gene transcription is regulated, making this P19 system a significant new model to study the physiological regulation of PKIs.
The three PKI protein isoforms – PKIα, PKIβ, and PKIγ– are produced from three evolutionarily conserved genes that have widespread but distinctive tissue distributions (Collins and Uhler 1997; Zheng et al. 2000). Mice deficient in PKIα exhibited defects in skeletal muscle, but show no gross defects in development or fertility (Gangolli et al. 2000). PKIβ-deficient mice exhibited a partial loss of PKI activity in testis, but remained fertile with normal testis development and function (Gangolli et al. 2000). However, detailed studies of neuronal development in the PKIβ deficient mice have not been carried out. Remarkably, PKIα/β double-knockout mice were also viable and fertile with no additional physiological defects (Belyamani et al. 2001). Mice deficient in PKIγ have not been described to date, and it is possible that PKIγ compensates to some extent for loss of PKIα and PKIβ in the mice deficient for the latter PKI isoforms. More recently, studies of osteosarcoma cells and fibroblasts have demonstrated that PKIγ is necessary for the efficient termination of PKA signaling in the nucleus (Chen et al. 2005).
Studies indicate that multiple forms of PKIβ exist, related by covalent modification and alternate translational initiation (Van Patten et al. 1991, 1997; Zheng et al. 2000; Kumar and Walsh 2002). PKIβ was first isolated from rat testis as a 70 amino acid protein, but the genomic sequence suggested that an alternate form might exist, arising as a consequence of alternate translational initiation. This species, now termed PKIβ78, is equipotent with PKIβ70, and also occurs in vivo. Six additional species of PKIβ are also evident in tissues: two of these represent the phospho forms of PKIβ78 and PKIβ70, while the other four represent phospho and dephospho forms of two higher molecular mass PKIβ species. These latter forms are currently termed PKIβ109 and PKIβY, and their molecular identities have yet to be fully determined (Kumar et al. 1997). Our data indicate that the form expressed in P19 cells corresponds to the 78 amino acid isoform of PKIβ expressed in the brain (Kumar et al. 1997). Furthermore, the gene organization of PKIβ elucidated from our RACE studies indicates that the P19 cell PKIβ78 is a specific inhibitor of PKA. Other isoforms of PKIβ exist that are dual-specificity inhibitors of both PKA and PKG, but the sequences required for PKG inhibition are located in exon 7, a region that is absent in the cDNA of PKIβ in P19 cells. Human PKIB shares a 70% homology to mouse PKIβ, most notably within the sequences for the pseudosubstrate site and nuclear export signal. In humans, PKIB is the predominant isoform expressed in the brain, and the PKIB cDNA also encodes a peptide of 78 amino acids (Zheng et al. 2000). Because of the sequence homology between human and mouse PKIβ and similar patterns of tissue specific expression, it is possible that PKIβ may play a role in human as well.
Although the tissue-specific expression of mammalian PKI genes has been well characterized in past studies, tissue-specific functions of the PKIs have not been described in detail previously. The findings reported here suggest that PKIβ can be highly regulated by bHLH proteins such as Ascl1 and that PKIβ has an isoform-specific role in the neuronal differentiation of P19 cells. More detailed studies in the P19 cells and in PKIβ-deficient animals should provide greater clarity into the properties of PKIβ that are important for neuronal differentiation.
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Figure S1. Organization of the PKIβ gene in P19 cells. (a) PKIβ gene organization was determined using the Smart™ RACE cDNA amplification kit, and a representative gel of the amplified products is shown. (b) Representative PKIβ nucleotide sequence from an isolated cDNA clone. The beginning of each exon of the PKIβ gene is underlined.
Table S1. shRNA sequences.
Table S2. Oligonucleotides used in the experimental methods.
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