Inhibition of PKCε induces primordial germ cell reprogramming into pluripotency by HIF1&2 upregulation and histone acetylation

Abstract

Historically, primordial germ cells (PGCs) have been a good model to study pluripotency. Despite their low numbers and limited accessibility in the mouse embryo, they can be easily and rapidly reprogrammed at high efficiency with external physicochemical factors and do not require transcription factor transfection. Employing this model to deepen our understanding of cell reprogramming, we specifically aimed to determine the relevance of Ca²⁺ signal transduction pathway components in the reprogramming process. Our results showed that PGC reprogramming requires a normal extracellular [Ca²⁺] range, in contrast to neoplastic or transformed cells, which can continue to proliferate in Ca²⁺-deficient media, differentiating normal reprogramming from neoplastic transformation. Our results also showed that a spike in extracellular [Ca²⁺] of 1-3 mM can directly reprogram PGC. Intracellular manipulation of Ca²⁺ signal transduction pathway components revealed that inhibition of classical Ca²⁺ and diacylglycerol (DAG)-dependent PKCs, or intriguingly, of only the novel DAG-dependent PKC, PKCε, were able to induce reprogramming. PKCε inhibition changed the metabolism of PGCs toward glycolysis, increasing the proportion of inactive mitochondria. This metabolic switch from oxidative phosphorylation to glycolysis is mediated by hypoxia-inducible factors (HIFs), given we found upregulation of both HIF1α and HIF2α in the first 48 hours of culturing. PKCε inhibition did not change the classical pluripotency gene expression of PGCs, Oct4, or Nanog. PKCε inhibition changed the histone acetylation of PGCs, with histones H2B, H3, and H4 becoming acetylated in PKCε-inhibited cultures (markers were H2BacK20, H3acK9, and H4acK5K8, K12, K16), suggesting that reprogramming by PKCε inhibition is mediated by histone acetylation.

Keywords: Cellular reprograming; HIF; PKC; calcium signaling; histone acetylation; pluripotency; primordial germ cells.

Figure 1

Number of PGCs (A, C, E, G) and reprogrammed EGCs (B, D, F, H) under various culture conditions. (A) Extracellular Ca²⁺ dose response in reprogramming condition cultures. At least 1 mM concentration is needed for PGC survival; (B) A minimum of 2 mM [Ca²⁺] is needed to allow for PGC reprogramming; (C) Extracellular Ca²⁺ dose response in non-reprogramming conditions. Again, at least 1 mM concentration is needed for PGC survival; (D) 1 mM to 3 mM [Ca²⁺] is enough to reprogram PGCs in non-reprogramming conditions (absence of FGF2); (E) No differences were found in PGC numbers in the presence of ionomycin, demonstrating it is neither toxic nor induces augmented proliferation in cultures at this concentration; (F) Ionomycin prevents PGC reprogramming by a statistically significant reduction in reprogrammed EGC colonies; (G) No differences were found in PGC numbers by the addition of flunarizine, demonstrating that it is neither toxic nor induces augmented proliferation in cultures at this concentration; (H) Flunarizine does not have an effect on PGC reprogramming. An asterisk represents a statistically significant p value < .05.

Figure 2

Number of PGCs (A, C, E, G) and reprogrammed EGCs (B, D, F, H) under non-reprogramming conditions (except for G&H) and different pharmacological supplementation. (A, C, E, G) No differences were found in PGC numbers in the presence of staurosporine, Ro-31-8220, PIP, and FR236924, demonstrating that these compounds are neither toxic nor induce augmented proliferation in cultures at the concentrations employed. (B) Staurosporine alone induces PGC reprogramming, by a statistically significant increase in reprogrammed EGC colonies. (D) Ro-31-8220 alone induces PGC reprogramming, by a statistically significant increase in reprogrammed EGC colonies. (F) PIP alone induces PGC reprogramming, by a statistically significant increase in reprogrammed EGC colonies. (H) FR236924 does not have an effect on PGC reprogramming under hypoxic conditions. An asterisk represents a statistically significant p value < .05.

Figure 3

EB formation and spontaneous differentiation of reprogrammed PGCs by PIP exposure. The markers for endoderm, ectoderm, and mesoderm are, respectively, albumin, AE1/AE3 cytokeratins, and vimentin. DAPI (blue) shows every cell nucleus. Scale bars correspond to 25 μm.

Figure 4

Flow cytometry data of PGC cultures showing the percentage of SSEA1⁺ cells displaying a green signal from the JC-1 probe (inactive mitochondria), a red signal (active mitochondria), or bivalent (both types) under non-reprogramming normoxia conditions (A), reprogramming hypoxia conditions (B), or PIP-supplemented cultures (C). An increase in inactive mitochondria to the detriment of bivalent mitochondria is observed in normoxia after 5 days of PIP supplementation (shown as percentage in D).

Figure 5

Confocal microscopy images for immunofluorescence against SSEA1 and HIF1α in PGC cultures. Samples include Cos7 cells cultured in normoxic conditions as a negative control and in hypoxia as a positive control. Images also show PGCs cultured in normoxia (Norm), hypoxia (Hyp), and PIP for 48 h. Note HIF1α expression in Cos7 and PGCs in hypoxia and in PIP-supplemented cultures. Scale bars correspond to 25 μm.

Figure 6

Confocal microscopy images for immunofluorescence against SSEA1 and HIF2α in PGC cultures. Samples include Cos7 cells cultured in normoxic conditions as a negative control and in hypoxia as a positive control. Images also show PGCs cultured in normoxia (norm), hypoxia (Hyp), and PIP for 48 h. Note HIF2 expression in Cos7 and PGCs in hypoxia and in PIP-supplemented cultures. Scale bars correspond to 25 μm.

Figure 7

Immunofluorescence microscopy against SSEA1 and c-Myc (A) and Klf4 (B) in PGC cultures, showing a negative reaction in SSEA1⁺ cell (PGCs) nuclei in PIP-supplemented cultures. Images also show PGCs cultured in normoxia (norm) and hypoxia (Hyp) for 48 h. Positive controls are the ESC line E14Tg2 and also hypoxic PGC cultures are positive for cMyc. Scale bars correspond to 25 µm.

Figure 8

Confocal merged images of H2BacK20, H3acK9, and H4acK5K8, K12, K16 immunofluorescence (in green in the nuclei) of PGC cultures under normoxic, hypoxic, and PIP conditions, showing a positive reaction in SSEA1⁺ cells (PGCs, membranes in red). Blue shows nuclei with DAPI stain. Colocalization is seen as light blue. Positive controls are an NIH3T3 mouse cell line subjected to the histone deacetylase inhibitor Na⁺ butyrate at 0.25 μM for 16 hours. Note histone acetylation in PGCs under reprogramming conditions only, both in hypoxia and PIP-supplemented cultures. Scale bars correspond to 100 μm in every photograph.