Cell Reprogram. 2015 Apr;17(2):95-105.
Prolonged proteasome inhibition cyclically upregulates Oct3/4 and Nanog gene expression, but reduces induced pluripotent stem cell colony formation.
- 1Ubiquitin Lab, Pennington Biomedical Research Center, Louisiana State University System , Baton Rouge, LA, 70803.
There is ample evidence that the ubiquitin-proteasome system is an important regulator of transcription and its activity is necessary for maintaining pluripotency and promoting cellular reprogramming. Moreover, proteasome activity contributes to maintaining the open chromatin structure found in pluripotent stem cells, acting as a transcriptional inhibitor at specific gene loci generally associated with differentiation. The current study was designed to understand further the role of proteasome inhibition in reprogramming and its ability to modulate endogenous expression of pluripotency-related genes and induced pluripotent stem cells (iPSCs) colony formation. Herein, we demonstrate that acute combinatorial treatment with the proteasome inhibitors MG101 or MG132 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) increases gene expression of the pluripotency marker Oct3/4, and that MG101 alone is as effective as VPA in the induction of Oct3/4 mRNA expression in fibroblasts. Prolonged proteasome inhibition cyclically upregulates gene expression of Oct3/4 and Nanog, but reduces colony formation in the presence of the iPSC induction cocktail. In conclusion, our results demonstrate that the 26S proteasome is an essential modulator in the reprogramming process. Its inhibition enhances expression of pluripotency-related genes; however, efficient colony formation requires proteasome activity. Therefore, discovery of small molecules that increase proteasome activity might lead to more efficient cell reprogramming and generation of pluripotent cells.
Selective degradation of intracellular proteins by the ubiquitin-proteasome system participates in cellular processes ranging from protein quality control to regulation of gene expression, cell cycle control, responses to oxidative stress and maintaining pluripotency. With regards to pluripotency, proteasome activity is associated with maintaining an open chromatin state and inhibiting expression of genes associated with differentiation. Several reports have demonstrated that inhibition of the proteasome using peptide aldehydes such as MG132 benefit early development of embryos generated by somatic cell nuclear transfer in a variety of species including mouse, bovine and porcine. Recently, our group reported that iPS cell colony formation improved 2-3 fold by treatment of cells with a histone deacetylase (HDAC) inhibitor prior to transfection with Yamanaka factors (OCT4, Sox2, cMyc, and Klf4). Based on these reports, we hypothesized that modulating proteasome activity using a proteasome inhibitor in combination with an HDAC inhibitor would impact the generation of induced pluripotent stem (iPS) cells.
Our report demonstrated that treatment with the proteasome inhibitor MG101, or MG132, in combination with the HDAC inhibitor valproic acid (VPA) increased Oct3/4 expression. Also, MG101 treatment of fibroblasts induced Oct3/4 gene expression and was as effective as VPA. Moreover, long term exposure of fibroblasts to low levels of MG101 or MG132 induced expression of Oct3/4 and nanog, but in a cyclical fashion. Surprisingly, long term exposure to proteasome inhibitors reduced colony formation. These results indicate that inhibiting proteasome activity can up-regulate genes such as Oct3/4. However, increasing expression levels of pluripotency genes is not sufficient for colony formation and that proteasome activity is required for somatic cell reprogramming.
This study is important for two reasons: 1) it contributes to our basic understanding of somatic cell reprogramming and 2) it suggests a possible path to further develop the technology for industrial scale applications. Specifically, our results indicate that the ubiquitin-proteasome system is linked to somatic cell reprogramming for iPS cell generation and that while inhibiting the 26S proteasome enhances pluripotent-related gene expression, colony formation requires proteasome activity. This suggests that proteasome regulation of genes like Oct3/4 or nanog does not necessarily correlate with pluripotency. Based on our results, the possibility exists that approaches aimed at increasing proteasome activity might translate into scalable methods to generate pluripotent cells (Figure 1).
Figure 1. Long term exposure to proteasome inhibitors reduce colony formation (1A). Approaches aimed at increasing proteasome activity might translate into scalable methods to generate pluripotent cells (1B).
Figure 2. Human foreskin fibroblasts were transduced with four transcription factors (Oct3/4, Sox2, Klf4 and c-Myc) by lentiviral transfection. Emerging colonies (A) were positive for pluripotency markers, e.g. alkaline phosphatase (B) and Sox-2 (C)
Elizabeth Floyd, Ph. D.
Kenneth J. Eilertsen, Ph. D.
Nuclear Reprogramming and Epigenetics Lab
Pennington Biomedical Research Center
6400 Perkins Road
Baton Rouge, LA 70808-4124