Comparative analysis of the human CPC cytoplasmic compartment
Whole label-free (LF) proteomic analyses of hCPC and hMSC [20] yielded 1,260 and 1,176 cytoplasmic proteins, respectively; 95% of which could be mapped onto a GO category. Ingenuity Pathway Analysis (IPA) of the cytoplasmic hCPC subproteome is shown (Fig. 1a). Cytoplasmic fractions of hCPC and hMSC were obtained and analyzed first by LF proteomics; 748 and 707 cytoplasmic proteins were identified in hCPC and hMSC, respectively (Supplementary Fig. S1 online). Among the cytoplasmic proteins expressed more differentially in hCPC, we identified 11 upregulated proteins, including 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2), procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1) and prolyl 4-hydroxylase, alpha polypeptide I/II, (P4HA1 and P4HA2) (Fig. 1b). We also identified 5 moderately downregulated proteins in hCPC, including aspartate beta-hydroxylase (ASPH) and insulin-like growth factor mRNA binding protein 2 (IGFBP2) (Fig. 1c). PANTHER GO-Slim analysis of biological processes using the upregulated hCPC cytoplasmatic proteins clearly indicated an over-representation of muscle contraction-associated proteins in hCPC (Fig. 1d) and PANTHER Pathway analysis showed an over-representation of cytoskeletal regulation by Rho GTPases (Supplementary Fig. S1 online).
To validate the proteomic data, we compared P4HA1 and ASPH expression in hCPC, hMSC and fibroblasts by RT-qPCR analysis. The data confirmed P4HA1 overexpression in hCPC (Fig. 1e). This was also confirmed by western blotting, although the difference in hCPC P4HA1 expression was less pronounced when compared with human fibroblasts, and no differences were evident when compared with hMSC (Fig. 1f). When we analyzed the gene expression of ASPH, we found that it was not downregulated in hCPC (Supplementary Fig. S1 online), as inferred from the proteomic analysis (Fig. 1e), suggesting a post-transcriptional regulation in hCPC. Additionally, we discarded the possibility that the expression differences found would be associated with the cardiac origin of the hCPC analyzed. We compared their diferential expression with total human heart samples confirming all results with the the sole exception of CDH5 (Supplementary Fig. S1 online).
Comparative analysis of the human CPC nuclear compartment
Analysis of the whole LF proteome in hCPC and hMSC yielded 446 and 514 nuclear proteins, respectively [20]; 95% of which could be included in a GO category. IPA analysis of the nuclear hCPC subproteome is shown (Fig. 2a). LF-proteomic analysis of purified nuclear fractions of hCPC and hMSC rendered 369 and 348 proteins, respectively (Supplementary Fig. S1 online). To confirm the proteins identified in the nuclear fraction, we compared with their representation in the cytoplasm (Fig. 2b,c). The comparative proteomic analysis of hCPC versus hMSC nuclear purified fractions revealed the potential differential expression of 27 proteins in hCPC (Fig. 2b,c). Of the more clearly overexpressed proteins in the hCPC nuclear compartment only IMP3 (also known as IGF2BP3), nestin and IL1A (Fig. 2b) also showed a parallel significant increase in mRNA expression in hCPC relative to hMSC. In addition, hCPC expressed lower nuclear levels of several proteins including Polymerase I and Transcript release factor (PTRF) (Fig. 2c); previous RNAseq studies [20] confirmed all dentified proteins. PANTHER GO-Slim analysis of biological processes using the upregulated hCPC nuclear proteins indicated a strong involvement in cellular component morphogenesis and organization, as well as muscle contraction (Fig. 2d). PANTHER Pathway analysis also revealed an important representation of the ubiquitin proteasome pathway (Supplementary Fig. S2 online).
Aiming to validate the proteomics nuclear data we evaluated the expression of ASPHD1 and PTRF, which were up- and down-regulated, respectively, in hCPC versus hMSC, by proteomics. RT-qPCR analysis confirmed a significant differential expression of ASPHD1 in all hCPC isolates (hCPC1–3) in comparison with hMSC and fibroblasts (Fig. 2e). The preferential expression in hCPC was also confirmed by western blotting and by immunofluorescence (Fig. 2f,g). By contrast, PTRF downregulation in hCPC was not confirmed by RT-qPCR (Supplementary Fig. S2 online), suggesting again a relevant post-transcriptional regulation.
Because a very significant fraction of regulatory nuclear proteins is expressed at low levels, below the detection limits of proteomics, we validated by RT-qPCR several transcriptional factors found up- and down-regulated, by RNAseq in hCPC compared with hMSC [20]. GATA4, SOX17, WT1 and GATA2 were robustly overexpressed in hCPC in comparison with hMSC (Supplementary Fig. S2 online). The expression levels of TBX3 and MEF2C were also significantly higher in hCPC than in hMSC, but less pronounced. We also confirmed that HOXD8 and HOXA10 were barely expressed in hCPC in comparison with hMSC (Supplementary Fig. S2 online).
IL1A is a dual-function cytokine in hCPC
Two of the most differentially expressed genes in hCPC were IL1A (as mentioned above) and IL1B, whose overexpressions were also validated by RNAseq [20]. IL1A was found over-represented by comparative LF-proteomic analysis in the nuclear compartment of hCPC compared with hMSC (Fig. 2c). IL1A is produced as a precursor protein that yields a mature form and an N-terminal propeptide, containing a nuclear localization sequence that allows access to the nuclear compartment. In this way, IL1A is a well established “dual-function cytokine” that plays a role in the nucleus independently of its classical extracellular mediated effects [25-27].
To validate the proteomic analysis, we assessed the expression of IL1A and IL1B by RT-qPCR in hCPC and hMSC. The results clearly confirmed the overexpression of IL1A and IL1B in hCPC (6,788- and 1,409-fold, respectively, Fig. 3a). We also tested the expression of other members of the IL1 signaling pathway. A significant increase (3.22-fold change) was found for the expression of the natural antagonist of IL1R1 (IL1RA) (Fig. 3a). Other main members of the IL1 pathway, IL1 receptor (IL1RI), IL38 and the secondary IL1 receptor (IL1R2) were, or not differentially expressed (IL1RI) or not detected. Immunofluorescence analysis of IL1A, with an antibody against the full size protein, revealed a highly preferential cytoplasmic location in basal conditions (homeostasis; Fig. 3b). However, while hCPC showed significantly higher levels of IL1A mRNA expression than hMSC (Fig. 3a), the latter showed higher levels of IL1A protein (Fig. 3b), suggesting an important lineage-specific post-transcriptional regulation. We thus obtained nuclear and cytoplasmic fractions from hCPC and analyzed IL1RI, IL1A and IL1B expression by western blotting. The results comfirmed an almost exclusive cytoplasmic location for all proteins analyzed (Supplementary Fig. S3 online). The fraction of nuclear IL1A detected by proteomics (Fig. 2b) thus seems to be minor. Given the known immunoregulatory capacity of hCPC [17-19] and their definition as an MSC-like cell subpopulation [19], it is possible that IL1A could play a role in hCPC in homeostasis. We thus evaluated whether IL1A could contribute to the immunoregulatory capacity of hCPC. Thus, we co-cultivated phytohemagglutinin-stimulated human CD3 T cells with control hCPC, hCPC silenced for the expression of IL1A (hCPC siIL1A), or negative-control tranfected cells (hCPC siNeg). We first confirmed that the siIL1A (10 nM) could downregulate IL1A (~72%) in hCPC (Supplementary Fig. S3 online). All cell populations (hCPC, hCPC siIL1A and hCPC siNeg) demonstrated similar immunoregulatory capacity at the higher cell doses analyzed (1:10–1:20), which was lost when lower doses were evaluated (1:40). Therefore no significant changes in immunoregulatory capacity were found (Fig. 3c), indicating that IL1A seems not to have a relevant role in the T cell immunoregulatory capacity of hCPC.
We next evaluated whether IL1A could acts as a dual-function cytokine in hCPC, as has previously been described in other cell lineages [28]. We analyzed in hCPC the behavior of IL1A, IL1B and IL1RI expression in response to oxidative stress (see Methods). We found that IL1RI expression was equivalently and moderately reduced (~25%) by both treatments (preferential apoptosis or necrosis). IL1Aand IL1B expression were also decreased by both treatments, but to a much greater extent; IL1A expression was more pronouncedly reduced (90–95% reduction) when necrosis was induced (Fig. 3d). Of note, IL1A behaved differently to the two stmuli in hCPC and hMSC, as evaluated by immunofluorescence. Upon induction of apoptosis, IL1A protein was significantly upregulated in hCPC, whereas it was clearly reduced in hMSC (Fig. 3e); induction of necrosis provoked a major loss of IL1A in both cell types (Fig. 3e). Quantification of nuclear versus cytoplasmic localization by immunofluorescence of IL1A in hCPC, comparing homeostasis with the induction of apoptosis or necrosis, revealed a significant increase in the nuclear location of IL1A (co-localization with DAPI signal) after the induction of apoptosis (Fig. 3f). The opposite was found in hMSC where co-localization of IL1A with the nuclear compartment was poorer upon apoptosis induction, although it was augmented after necrosis induction (Fig. 3f). We also performed western blotting of subcellular compartments in hCPC subjected to apoptosis (Fig. 3g); in necrotic cells, expression of the three proteins was quite low and difficult to quantify because of the strong loss of cellular content. In agreement with the immunofluorescence study, a substantial fraction of IL1A and IL1B was found in the nuclear compartment (Fig. 3g) whereas the subcellular localization of IL1RI was barely unchanged by the induction of apoptosis (compare with Supplementary Fig. S3 online).
Comparison of nuclear/cytoplasmic ratio in apoptosis versus homeostasis, in a representative western experiment, yielded an important increment for IL1A and IL1B (IL1A> IL1B), compared with a modest variation on IL1RI (Fig. 3g). Altogether, these data suggest that IL1A could be acting as dual-cytokine in hCPC with a potential role in the transcriptional regulation in apoptosis.
Functional evaluation of IMP3 in hCPC in homeostasis and in response to oxidative damage
IGF2 is the predominant form of IGF in humans [29] and it binds to insulin-like growth factor 1 receptor (IGF1R), insulin-like growth factor 2 receptor (IGF2R; CD222) and the insulin receptor A isoform (IR-A). It seemed interesting that in addition to IGF2R, two additional members of the IGF2 pathway (insulin-like growth factor mRNA binding proteins 2 and 3; IMP2/IMP3) were identified as over-represented in the hCPC nuclear subproteome by LF-proteomics (Fig. 2b). RT-qPCR analysis validated the high levels of IMP3 expression in hCPC versus hMSC (>40-fold overexpression), but the opposite was observed for IMP2 (Fig. 4a).
IMP3 belongs to a family of mRNA-binding proteins that bind to multiple mRNAs in mammalian cells, including IGF2 [30]. Based on previous literature [29], we hypothesized that high levels of IMP3 would lead to a decrease in the autocrine bioavailability of IGF2, reducing the potential signaling through IGF2R and triggering senescence/apoptosis. We first analyzed the impact of IMP3 knockdown in two independent hCPC isolates. Cells transfected with siIMP3 showed significantly reduced levels of IMP3 when compared with negative control or non-transfected control cells, analyzed both by RT-qPCR and western blotting (Fig. 4b,c). IMP3 silencing did not affect negativelly hCPC viability, 48 h post-transfection; in fact, IMP3-silenced cells showed a moderate increase in viability (Fig. 4d). We then analyzed the effects of IMP3 silencing on the response of hCPC to oxidative stress (500 mM H2O2, during 48 h) and evaluating apoptotic and necrotic cells with the Annexin V/propidium iodide (Supplementary Fig. S3 online). Neither of the two hCPC populations tested showed any remarkable difference in the percentages of homeostatic, apoptotic, late apoptotic or necrotic cells (Fig. 4e). Thus, IMP3 does not seem to play a critical role in the regulation of hCPC response to oxidative stress-mediated apoptosis.
Although IMP3 seems not to be essential for apoptotic responses we investigated IMP3 regulation in hCPC damage responses. We first studied the impact of apoptosis or necrosis induction on the transcriptional activity of IMP3, IMP2 and IGF2R, and their subcellular localization. Neither apopotosis nor necrosis affected IGF2R expression; however, the induction of apoptosis (but not necrosis) promoted a significant decrease of IMP3 and IMP2 transcription in hCPC (Fig. 5a). Western blotting of hCPC showed that IGF2R and IMP3 were expressed at similar levels whereas IMP2 was expressed at apparently lower levels (Fig. 5b). Analysis of nuclear and cytoplasmic fractions by western blotting confirmed that a substantial fraction of IMP3 (22–40%), and to a lesser extent IMP2, was found in the nuclear fraction in homeostasis (Fig. 5b, left panel). After induction of apoptosis, both IMP2 and IMP3 showed an increased presence in the nuclear compartment with respect to the cytoplasmic compartment, whereas IGF2R was unchanged (Fig. 5b; right panel); specifically, we found an increase in nuclear IMP2 and IMP3 of 8.5-fold and 13-fold, respectively (Fig, 5c). These results were confirmed by immunofluorescence (Fig. 5d). We analyzed the nuclear versus cytoplasmic localization of the IMP3 fluorescent signal and we confirmed that, upon induction of apoptosis, the nuclear pool of IMP3 singnificantly increases (co-localization coefficient referred to DAPI signal) (Fig. 5d).
Thus, apoptosis induction in hCPC triggers a significant decrease in IMP2 and IMP3 transcription, concomitant with an enrichement of both proteins in the nuclear compartment. These results suggest a non-essential role of IMP3 in gene expression regulation upon induction of oxidative stress-mediated apoptosis.
IMP3 regulates proliferation and migration of hCPC
Analysis of proliferation in IMP3-silenced cells and controls estimated by EdU (5-ethynyl-2’-deoxyuridine) incorporation during 12 h and 48 h post-transfection revealed that the knockdown of IMP3 significantly reduced proliferation in hCPC1 cells (about 2-fold) and a similar effect was found in hCPC3 cells (Fig. 6a,b). Thus, IMP3 seems not to be relevant for survival, but is likely involved in hCPC proliferation regulation.
We then evaluated the potential implication of IMP3 in cell motility, as previously proposed [31] using wound-healing assays. Monolayers of hCPC cells silenced or not for IMP3 were compared in their capacity to repair a wound during 24 h, as described [20]. As shown in Fig. 6c, both IMP3-silenced hCPC isolates demonstrated a statistically significant delay (at 9–12 h) in wound healing, albeit with different kinetics (Fig. 6c).
Finally, we analyzed a panel of candidate target genes previously reported to be regulated by IMP3 in heterologous models, or described as preferentially expressed in hCPC [20, 22]. We used RT-qPCR to compare the levels of gene expression in hCPC silenced for IMP3 knockdown or in control cells. Figure 6d shows the results obtained with isolate hCPC1, which showed more robust IMP3 silencing.
Concerning genes involved in proliferation, we found that PTRF (also known as Cavin1 or Cavin-1) and HMGA2 (high-mobility group AT-hook 2) were significantly downregulated in siIMP3 cells (~50%). c-MYC, CDK6 and CD9 were moderately but not significantly downregulated (<20%). In relation to genes involved in apoptosis, we found that ICAM3 expression was significantly reduced (~60%) in IMP3-silenced cells, but unexpectedly not NEMO (inhibitor of nuclear factor kappa B kinase subunit gamma).
We also tested the consequence of IMP3 silencing for the expression of a small panel of transcriptional factors previously (Supplementary Fig. S2 online) defined in hCPC. All them, except SOX17 expression (~40% reduction), did not modify the expression after IMP3 knockdown. These results suggest that IMP3 might have a modest role in regulating hCPC fate-genes by regulation of SOX17. We additionally analyzed the potential impact of IMP3 knockdown on several genes associated in other cell types with self-renewal, such as Oct4, Dido3 and Mbd3, [20, 22, 32]. The expression of all three genes was unaffected by IMP3 silencing (Fig. 6d). These results suggest that IMP3 seems not to be mainly involved in the regulation of the undifferentiated state of hCPC. A similar analysis using hCPC3 yielded essentially identical but non-significant results.
Finally, because nuclear IL1A also regulates cell proliferation and migration [26, 27], we sought to evaluate the potential involvement of IMP3 in the regulation of the IL1 pathway. IMP3-silenced hCPC and controls were evaluated by RT-qPCR 48 h post-transfection. Compared with control hCPC, IMP3-silencing failed to affect IL1A expression but enhanced IL1B expression (Supplementary Fig. S3 online). IL1RA and IL1R1 were also unaffected by IMP3-silencing (Supplementary Fig. S3 online), indicating that IMP3 is likely not involved in the regulation of IL1 pathway in hCPC.