Dickey-Sims C et al. (2005), Runx-dependent expression of PKC is critical fo...

ECB-ART-39515

BMC Biol 2005 Aug 02;3:18. doi: 10.1186/1741-7007-3-18.

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Runx-dependent expression of PKC is critical for cell survival in the sea urchin embryo.

Dickey-Sims C , Robertson AJ , Rupp DE , McCarthy JJ , Coffman JA .

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BACKGROUND: Runx transcription factors play critical roles in the developmental control of cell fate and contribute variously as oncoproteins and tumor suppressors to leukemia and other cancers. To discover fundamental Runx functions in the cell biology of animal development, we have employed morpholino antisense-mediated knockdown of the sea urchin Runx protein SpRunt-1. Previously we showed that embryos depleted of SpRunt-1 arrest development at early gastrula stage and underexpress the conventional protein kinase C SpPKC1. RESULTS: We report here that SpRunt-1 deficiency leads to ectopic cell proliferation and extensive apoptosis. Suppression of the apoptosis by pharmacological inhibition of caspase-3 prevents the ectopic proliferation and rescues gastrulation, indicating that many of the overt defects obtained by knockdown of SpRunt-1 are secondary to the apoptosis. Inhibition or knockdown of SpPKC1 also causes apoptosis, while cell survival is rescued in SpRunt-1 morphant embryos coinjected with SpPKC1 mRNA, suggesting that the apoptosis associated with SpRunt-1 deficiency is caused by the deficit in SpPKC1 expression. Chromatin immunoprecipitation indicates that SpRunt-1 interacts physically with SpPKC1 in vivo, and cis-regulatory analysis shows that this interaction activates SpPKC1 transcription. CONCLUSIONS: Our results show that Runx-dependent activation of SpPKC1 is essential for maintaining protein kinase C activity at levels conducive to cell survival during embryogenesis.

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Species referenced: Echinodermata
Genes referenced: CyIIIa LOC100887844 LOC115919910 LOC583082 LOC586799 LOC587333 pkcl2 runx2
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	Figure 1. Morpholino antisense-mediated knockdown of SpRunt-1 leads to ectopic proliferation and apoptosis in the gastrula stage (48 h) embryo. (A-B) Differential interference contrast (DIC) images of embryos injected with (A) control MASO and (B) SpRunt-1 MASO. (C-D) Confocal micrographs (single optical sections) of BrdU-labeled embryos injected with (C) control MASO and (D) SpRunt-1 MASO. (E-F) Confocal sections of TUNEL (green)- and DAPI (blue, DNA stain)-labeled embryos injected with (E) control MASO and (F) SpRunt-1 MASO. (G-H) Confocal sections of antiactivated caspase-3 (green)- and DAPI (blue)-labeled embryos injected with (G) control MASO and (H) SpRunt-1 MASO. Scale bar = 20 μm.
	Figure 2. Cell survival in SpRunt-1 morphant embryos is rescued by either injection of full-length SpRunt-1 mRNA, treatment with a caspase-3 inhibitor or injection of exogenous SpPKC1 mRNA. (A) DIC image of 48 h SpRunt-1 morphant embryo. (B) Confocal section of 48 h SpRunt-1 morphant embryo labeled with DAPI (blue) and TUNEL (green). (C) Confocal section of 48 h SpRunt-1 morphant embryo that was coinjected with full-length SpRunt-1 mRNA and then labeled with DAPI and TUNEL as in (B). All of the imaged embryos (6/6) from this group had the TUNEL-negative phenotype shown, and 12/12 imaged embryos in a separate rescue experiment were similarly TUNEL-negative. (D) DIC image of 48 h SpRunt-1 morphant embryo, from same set of injected embryos as that shown in (A) but treated with the caspase-3 inhibitor Ac-DEVD-CHO. (E) Confocal section of 48 h SpRunt-1 morphant embryo treated with Ac-DEVD-CHO and then labeled with DAPI and TUNEL as in (B). In a separate experiment, the frequency of imaged Ac-DEVD-CHO-treated morphants displaying a TUNEL-negative phenotype similar to that shown here was 11/21, similar to the frequency of morphants displaying rescued gastrulation (Table 1). (F) DIC image of 48 h embryo injected with SpRunt-1 MASO + SpPKC1 mRNA. (G) Confocal section of 48 h embryo injected with SpRunt-1 MASO + SpPKC1 mRNA and then labeled with DAPI and TUNEL as in (B). Arrows point to invaginated guts, which are outlined in (C), (E) and (G).
	Figure 3. Inhibition or knockdown of PKC leads to apoptosis that is suppressed by inhibition of caspase-3. (A-E) DIC images, (F-J) fluorescent confocal images of embryos subjected to TUNEL (green) and counterstained with DAPI (blue) and (K-O) immunofluorescent confocal images of embryos stained for activated caspase-3 (green) and counterstained with DAPI (blue). (A), (F) and (K) are control embryos; (B), (G) and (L) are embryos treated with the PKC inhibitor chelerythrine from mesenchyme blastula stage; (C), (H) and (M) are embryos treated with chelerythrine and the caspase-3 inhibitor Ac-DEVD-CHO; (D), (I) and (N) are embryos injected with a SpPKC1 MASO; (E), (J) and (O) are embryos injected with the SpPKC1 MASO and treated with Ac-DEVD-CHO. The high levels of activated caspase-3 staining in panels (M) and (O) suggest that caspase-3 has been processed as a result of initiation of the apoptotic program but remains proteolytically inactive because of the presence of the inhibitor.
	Figure 4. SpPKC1 is a direct target of SpRunt-1. (A) Schematic of the 5' flanking sequence of SpPKC1, showing locations and sequences of the Runx consensus binding elements (underlined bases mutated for reporter gene studies). (B) EMSA of recombinant Runt domain (RD)-CBFβ heterodimers using probes consisting of target sequences from CyIIIa [12] and the distal and proximal Runx sites from SpPKC1. A 100-fold excess of unlabeled CyIIIa sequence was used as a competitor to test the specificity of the interactions. "C", complex between Runt domain and probe; "FP", free probe. (C) Chromatin immunoprecipitation (ChIP) from 48 h embryos using either an anti-SpRunt-1 [12] or nonimmune IgG. CyIIIa serves as a positive control, whereas histone H4 sequences (present as several hundred copies per haploid genome) serves as a negative control for specificity. (D) Relative normalized expression of wild-type (WT) PKC1-luc and mutant PKC1-luc, in which the proximal and distal Runx target sites have been mutated individually or in combination to abolish SpRunt-1 binding (PM, DM, and PM+DM, respectively), expression in control embryos and embryos injected with SpRunt-1 MASO. Fold difference in expression levels of each construct and of the basal luciferase vector alone (V) compared to the WT construct was measured by quantitative RT-PCR [38]. Each bar represents the averages and standard deviations from three to five different experiments. (E) Pathway summarizing the experimental approaches and results showing that SpRunt-1 promotes cell survival via transcriptional activation of SpPKC1.

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