REX-1 expression and p38 MAPK activation status can determine proliferation/differentiation fates in human mesenchymal stem cells

Abstract

Background: REX1/ZFP42 is a well-known embryonic stem cell (ESC) marker. However, the role of REX1, itself, is relatively unknown because the function of REX1 has only been reported in the differentiation of ESCs via STAT signaling pathways. Human mesenchymal stem cells (hMSCs) isolated from young tissues and cancer cells express REX1.

Methodology/principal finding: Human umbilical cord blood-derived MSCs (hUCB-MSCs) and adipose tissue-derived MSCs (hAD-MSCs) strongly express REX1 and have a lower activation status of p38 MAPK, but bone marrow-derived MSCs (hBM-MSCs) have weak REX1 expression and higher activation of p38 MAPK. These results indicated that REX1 expression in hMSCs was positively correlated with proliferation rates but inversely correlated with the phosphorylation of p38 MAPK. In hUCB-MSCs, the roles of REX1 and p38 MAPK were investigated, and a knockdown study was performed using a lentiviral vector-based small hairpin RNA (shRNA). After REX1 knockdown, decreased cell proliferation was observed. In REX1 knocked-down hUCB-MSCs, the osteogenic differentiation ability deteriorated, but the adipogenic potential increased or was similar to that observed in the controls. The phosphorylation of p38 MAPK in hUCB-MSCs significantly increased after REX1 knockdown. After p38 MAPK inhibitor treatment, the cell growth in REX1 knocked-down hUCB-MSCs almost recovered, and the suppressed expression levels of CDK2 and CCND1 were also restored. The expression of MKK3, an upstream regulator of p38 MAPK, significantly increased in REX1 knocked-down hUCB-MSCs. The direct binding of REX1 to the MKK3 gene was confirmed by a chromatin immunoprecipitation (ChIP) assay.

Conclusions/significance: These findings showed that REX1 regulates the proliferation/differentiation of hMSCs through the suppression of p38 MAPK signaling via the direct suppression of MKK3. Therefore, p38 MAPK and REX-1 status can determine the cell fate of adult stem cells (ASCs). These results were the first to show the role of REX1 in the proliferation/differentiation of ASCs.

Figure 1. The expression levels of REX1, p38 MAPK and the cell proliferation of hMSCs.

(A) The expression of REX1 in hUCB-MSCs, hAD-MSCs and hBM-MSCs. hUCB-MSCs and hAD-MSCs have strong REX1 expression, but hBM-MSCs have very weak REX1 expression. (B) The expression of p38 and phosphorylated p38 (Pp38) in hUCB-MSCs, hAD-MSCs and hBM-MSCs. The expression level of p38 was similar in the three types of hMSCs examined. hBM-MSCs have the strongest expression level of Pp38 among the three types of hMSCs. (C) Cell proliferation measured by CCK-8. The growth of hBM-MSCs was slowest among the three types of hMSCs. (D) REX1 primarily localized in the nucleus of hUCB-MSCs. Scale bars represent 100 µm. Error bars represent the standard deviation from three independent experiments.

Figure 2. REX1 knockdown resulted in the growth retardation of hUCB-MSCs.

(A) The expression of REX1 decreased after REX1-knockdown lentivirus infection. (B) The cell proliferation of REX1 knocked-down hUCB-MSCs significantly decreased from two days after infection. (C) The morphology of cells three days after culturing. REX1 knocked-down cells are not growing at a rate similar to the control cells. (D) Cell cycle arrest was observed in REX1 knocked-down hUCB-MSCs using FACS analysis. (E) The expression levels of Cyclins, CDK and cell cycle inhibitors. The expression levels of CDK2, CCNB1, CDK4 and CCND1 decreased, but p27 did not change after REX1 knockdown. Error bars represent the standard deviation from three independent experiments. *, p<0.05; **, p<0.01.

Figure 3. The levels of p38 MAPK, Cyclins and cell cycle inhibitors changed after REX1 knockdown and p38 MAPK inhibitor treatment.

(A) Significant phosphorylation of p38 MAPK (Pp38) was seen after REX1 knockdown in hUCB-MSCs. (B) hUCB-MSCs, hAD-MSCs and hBM-MSCs express all four types of p38 isoforms. (C) After p38 MAPK inhibitor treatment (1 µM BIRB796), the phosphorylation of p38 MAPK was similar in REX1 knocked-down hUCB-MSCs compared to vehicle control-infected hUCB-MSCs. (D) Cell proliferation was measured with CCK-8 for four days. The cell proliferation of REX1 knocked-down hUCB-MSCs was similar with vehicle control-infected hUCB-MSCs after BIRB796 (1 µM) treatment. Without BIRB796, the proliferation of REX1 knocked-down hUCB-MSCs significantly decreased compared with vehicle control-infected hUCB-MSCs. **, p<0.01. (E) The p38 MAPK of REX1 knocked-down hUCB-MSCs was not activated after 1 µM or 10 µM BIRB796 treatment compared to those of vehicle control-infected hUCB-MSCs. (F) Changes in the expression of CDK and cell cycle inhibitors. The decreased expression of CDK2 and CCND1 recovered after p38 MAPK inhibitor treatment. (G) The expression levels of ERK1/2, MEK, phospho-MEK (pMEK1/2) and NF-κB did not significantly change after REX1 knockdown in hUCB-MSCs. (H) The expression changes of STAT3 and STAT5. The expression levels of STAT3 and phospho-STAT3 were significantly increased after REX1 knockdown without regard to p38 MAPK inhibitor treatment. STAT5 expression did not change after the knockdown of REX1. (I) p38α and p38β were up-regulated but p38γ was down-regulated after REX1 knockdown in hUCB-MSCs. (J) REX1 expression of hUCB-MSCs did not change after BIRB796 (p38 MAPK inhibitor) treatment. Error bars represent the standard deviation from three independent experiments.

Figure 4. The expression of MKK3/6 in REX1 knocked-down hUCB-MSCs and normally cultured hMSCs, and the MKK3 ChIP assay in hUCB-MSCs.

(A) MKK3 expression significantly increased, but MKK6 did not significantly change after REX1 knockdown as shown by RT-PCR. (B) MKK3 increased after REX1 knockdown at the protein expression level. (C) The expression level of MKK3 in hUCB-MSCs was 20-fold less than the level in hBM-MSCs under normal culture conditions as shown by real-time RT-PCR. (D) The ChIP assay for REX1. Three regions have REX1 consensus sequences in the MKK3 genomic DNA. REX1 binds to the first exon region (MKK3Exon1) of MKK3. Abbreviations: MKK3Promt, MKK3 promoter region; MKK3Exon1, MKK3 exon 1 region; MKK3Intron1, MKK3 intron 1 region. Error bars represent the standard deviation from three independent experiments.

Figure 5. Differentiation study, NOTCH and WNT expression changes in REX1 knocked-down hUCB-MSCs.

(A) After a three week induction, adipogenic and osteogenic differentiated hUCB-MSCs were stained with Oil Red O or Alizarin Red S. The number of adipogenic differentiated cells was similar to vehicle control-infected hUCB-MSCs or was slightly increased in REX1 knocked-down hUCB-MSCs. Osteogenesis of REX1 knocked-down hUCB-MSCs decreased compared to vehicle control-infected hUCB-MSCs. (B) The expression changes of NOTCH signaling genes after REX1 knockdown. The expression levels of JAG1, NOTCH1 and NOTCH4 increased after REX1 knockdown. HES1 expression increased 1.2-fold after REX1 knockdown. (C) The expression levels of GSK3-β, phospho-GSK3-β at serine-9 (pGSK3β) and β-CATENIN decreased after REX1 knock-down in hUCB-MSCs. (D) The expression levels of FZD2, LRP5 and DKK1 decreased after REX1 knockdown in hUCB-MSCs. (E) The expression levels of core transcription factors and polycomb group genes. The expression level of ZNF281 was down-regulated after REX1 knockdown in hUCB-MSCs. The expression levels of SUZ12 and BMI1 also decreased after REX1 knockdown. The expression of c-MYC did not change after REX1 knockdown. Abbreviations: Adipo, adipogenic induction; Osteo, osteogenic induction. Error bars represent the standard deviation from three independent experiments. Scale bars represent 100 µm.

Figure 6. Apoptosis after REX1 knockdown in hUCB-MSCs and REX1 inhibition in hMSCs and the summary of the role of REX1 in stem cells.

(A, B) Apoptotic cell death was not significantly different in REX1 knocked-down and vehicle control-infected hUCB-MSCs. (C) The expression of BAX did not change after REX1 knockdown in hUCB-MSCs. (D) Significant activation of p38 MAPK was observed after REX1 knockdown in hUCB-MSCs and hAD-MSCs but only slightly increased in hBM-MSCs. hBM-MSCs, which have low expression of REX1, have highly activated p38 MAPK (Pp38) in vehicle control-infected cells. (E) REX1 suppresses MKK3 expression, which activates p38 MAPK. REX1 also suppresses STAT3 expression and NOTCH signals. Error bars represent the standard deviation from three independent experiments.