Characterization of adipose-derived hMSCs by clonogenicity and molecular marker expression
The adipose-derived hMSCs used in this study were obtained from a commercial source (see “Methods”). Previously we have shown that these cells were able to differentiate into adipocytes and osteocytes in response to appropriate external stimuli [55]. To gain a better understanding of these cells, both clonogenicity of these cells as well as the expression of three known hMSCs markers, CD73, CD90 and CD105, were examined at passage 4 (P4), the same passage cells used in all subsequent experiments.
Of 3 independent repeats, the average clonogenicity (number of cells per clone is > 50) was determined to be 8% (± 0.67%) (Additional file 1: Figure S1).
To determine the expression of the three marker genes, immunostaining was carried out first. Both CD73 and CD105 were shown to be expressed almost ubiquitously, especially with CD105 whose expression in each individual cell can be easily discerned (Additional file 2: Figure S2, top 2 rows). Immunostaining with antibodies against CD90 from two different sources appeared to be challenging despite several attempts with various conditions, which had also been predicted due to indicated formalin sensitivity of epitope recognized by the antibody. Flow cytometry was subsequently used to analyze the expression of CD90. As control, cells were also co-stained with antibody against CD73. About 96 and 95.72% of cells independently express CD73 and CD90 respectively, and about 95% of cells co-express both (Additional file 2: Figure S2, bottom 2 rows).
In conclusion, the above results indicate that the adipose-derived hMSCs used in this study demonstrated 8% clonogenicity, with about 95% of them expressing all three markers, CD73, CD90 and CD105.
Temporal expression of RGS2 and RGS4 during adipogenesis and osteogenesis
Our initial interest in members of the regulator of G protein signaling (RGS) family began with a microarray analysis aimed at identifying novel regulators of human adipogenesis (unpublished data). Briefly, the expression profiles of hMSCs exposed to adipogenic differentiation condition (IBMX + DEX + insulin in growth media) was compared to undifferentiated hMSCs at 36 and 72 h post induction. Through this analysis, RGS2 and RGS4 were found to be significantly up- and down-regulated respectively during early adipogenesis, with RGS2 up regulated by 15-folds, and RGS4 down regulated by 100-folds at 72 h post induction. Because adipose and bone cells share common progenitor cells and there are shared regulators between adipogenesis and osteogenesis [17, 18], we expanded our interest in understanding the role of RGS2 and RGS4 in both adipogenesis and osteogenesis.
We first sought to determine the temporal expression pattern of RGS2 and RGS4 during both osteogenic and adipogenic differentiation of hMSCs. For adipogenesis, hMSCs were cultured in hyclone growth media (CM) supplemented with IBMX (0.45 µM), DEX (1 µM), and insulin (10 µg/ml), which is abbreviated as adipogenic inducing media (AIM). For osteogenesis, cells were cultured in CM supplemented with DEX (0.2 µM), β-glycerophosphate (10 mM), and ascorbic acid-2-phosphate (0.05 mM), which is abbreviated as osteogenic inducing media (OIM). DEX is a common component used in hMSC differentiation into adipocyte, osteocyte, and chondrocytes, and is thought to be necessary to potentiate differentiation and prevent apoptosis [56, 57]. Therefore, to also dissect the role of individual component of differentiation inducing cocktails on the expression of RGS2 and RGS4, RT-PCR was performed on both genes in hMSCs cultured in 8 different media treatments that include CM (control group), DEX (0.2 µM), DEX (1 µM), DEX (0.2 µM) + IBMX, AIM with 0.2 µM DEX, AIM with 1 µM DEX, OIM with 0.2 µM DEX, and OIM with 1 µM DEX, at eight different time points including D0.5, D1, D1.5, D2, D4, D5, D6 and D7 post initial treatment, with media change at 48-h intervals. All treatment group media was made in CM. Transcript level of RGS2 and RGS4 in each treatment group were normalized to that of a housekeeping gene HSP90 (internal control) and compared to its normalized level in CM control at corresponding time point.
As expected, expression of C/EBPα and PPARγ, two well-known master regulators for adipogenic lineage commitment, were both highly up regulated in AIM treated cells (Fig. 1a, b). Similarly, expression of LPL, a relatively later stage adipogenic marker encoding a lipoprotein lipase that breaks down lipids [58]. was also highly enriched in AIM treated cells (Fig. 1c) (Expression of LPL was undetectable in CM groups, so its expression in AIM and OIM treated cells was compared to its expression in DEX treated cells). On the other hand, expression of Runx2, a master regulator for osteogenic differentiation [14], was expressed at a higher level in OIM treated cells relative to AIM treated cells, especially after D4 (Fig. 1d). However, expression of osteocalcin (OC), which encodes a bone specific protein synthesized by osteoblast and serves as a marker of osteogenic maturation [59], appeared to be only slightly upregulated in DEX and OIM treated cells compared to its expression in AIM before D4, but reached to a similar level across all treatment groups thereafter (Fig. 1e).
Expression of RGS4 remained high in hMSCs cultured in CM throughout D0.5 to D7, but was differentially regulated in response to adipogenic induction (DEX + IBMX or AIM) vs. osteogenic induction (OIM) (Fig. 2a). Starting as early as D0.5, adipogenic induction by DEX + IBMX or AIM, regardless of DEX concentrations, resulted in down regulation of RGS4 by 2.5- to 5-folds. By D1, RGS4 was barely detectable and remained significantly down-regulated throughout the remaining treatment duration (Fig. 2). In OIM treatments, again regardless of DEX concentrations, RGS4 expression followed a similar pattern as in DEX treatment alone, first slightly downregulated by 1.5- to 3.3-folds between D0.5 and D2 and then upregulated by up to 3.75-fold between D4 and D7 (Fig. 2a). Overall, RGS4 expression is down regulated by all adipogenic treatment conditions starting as early as 12 h post treatment initiation, and upregulated by osteogenic treatments starting D4 in a pattern similar to DEX only treatment.
Expression of RGS2 is upregulated by all treatment types relative to its expression in control CM, though the degree of changes differs between adipogenic conditions and the other treatment conditions at certain time points (Fig. 2b). In contrast to RGS4, RGS2 expression was very low in hMSCs cultured in CM (Fig. 2b). Similar to RGS4 however, RGS2 expression in OIM followed the same pattern as its expression in DEX treatment alone, regardless of DEX concentrations. At D0.5, RGS2 was upregulated by 3.5- to 5-folds across all treatment groups compared to CM. At D1, RGS2 expression was induced to 12- to 14-folds higher in all adipogenic treatment conditions as compared to CM, which is about twice its level in DEX alone or OIM conditions. Between D1.5 and D4, its overall expression level was reduced across all treatment groups as compared to D1, but remained about twofold higher in all adipogenic conditions as compared to OIM or DEX alone. By D5, there was no significant difference across different treatment groups and by D6, RGS2 expression across all treatment groups dropped to similar levels as in CM control. Overall, expression of RGS2 is upregulated by all treatment types throughout D0.5 to D5, with significantly greater gain in adipogenic conditions (around twofolds) compared to the other treatment conditions between D1 and D4.
In conclusion, expression of both RGS2 and RGS4 in OIM treatment was regulated in parallel to that by dexamethasone treatment alone, regardless of DEX concentrations, indicating that the other two components in OIM media, AA-2-P and β-glycerophosphate, had no significant effect on RGS2 and RGS4 expression. RGS2 was upregulated by both DEX and OIM starting as early as D0.5 and subsiding by D6, whereas RGS4 was slightly downregulated by DEX and OIM during D0.5 to D2 but up-regulated afterwards. On the contrary, expression of both RGS2 and RGS4 differs significantly in adipogenic conditions as compared to in DEX alone, regardless of DEX concentration, indicating that IBMX and/or insulin exerted additional effect on their expression. Since their expression in IBMX + DEX inducing media is highly similar as in AIM, it suggests that IBMX, and not insulin, exerts such effect. Similar to DEX treatment, adipogenic treatment enhanced RGS2 expression until D6, but at significantly higher level (around twofolds) than DEX alone during D1 to D4. For RGS4 expression however, adipogenic treatment not only significantly inhibited the expression of RGS4 at a much greater level than DEX alone during D0.5 to D2 (near undetectable level), but also continued to downregulate its expression throughout the remaining course when it was being upregulated by DEX alone. Hence regulation of RGS2 and RGS4 expression were completely opposite to each other in adipogenic conditions.
Regulation of RGS2 and RGS4 during adipogenic and osteogenic differentiation is independent of media type
Since the Hyclone growth media (CM) used in composing the AIM media for adipogenic induction is a proprietary product that might contain unknown growth factor supplement, we wondered whether expression of RGS2 and RGS4 would remain similar in AIM based on a different growth media. Temporal expression pattern of RGS2 and RGS4 was then re-examined in parallel in culture conditions based on Hyclone CM, heat-inactivated fetal bovine serum (FBS) in DMEM complete media (HI-FBS CM), or FBS in DMEM complete media (FBS CM). The conditions included Hyclone CM or HI-FBS CM, Hyclone CM DEX (1 µM) or HI-FBS CM DEX (1 µM), Hyclone CM based AIM or HI-FBS CM based AIM or FBS CM based AIM (all with 1 µM DEX), and Hyclone CM based OIM (with 1 µM DEX) at D0.5, D1, D1.5, D2, D3 and D4 post adipogenic initiation (Additional file 3: Figure S3).
Similar to previous results, expression of RGS4 was downregulated by both DEX alone and AIM from D0.5 to D3, regardless whether they were Hyclone CM or HI-FBS CM based, however, its level was significantly higher in Hyclone CM based AIM than in HI-FBS CM based AIM at day 1 and day 2 (Additional file 3: Figure S3A). Similarly, RGS2 expression was upregulated by both DEX alone and AIM, regardless whether they were Hyclone CM or HI-FBS CM based. However, its surge in Hyclone CM based AIM was significantly greater (by up to twofolds) than that in HI-FBS CM based AIM (Additional file 3: Figure S3B). As a parallel control, expression of RGS2 and RGS4 in Hyclone OIM remain similar to previously described results.
In conclusion, the above results indicate that regulation of RGS2 and RGS4 during adipogenic differentiation is independent of media type, though the degree of change could vary. Next, we sought to determine the role of RGS4 and RGS2 during adipogenic and osteogenic differentiation through siRNA mediated gene silencing.
Expression knockdown of RGS2 and RGS4 in differentiating ad-hMSCs by reverse siRNA transfection
Previously, we identified XtremeGENE siRNA transfection reagent as a highly efficient siRNA delivery system in bone marrow derived hMSCs [17]. To confirm the effectiveness of this transfection reagent in adipose derived hMSCs (Ad-hMSCs), Ad-hMSCs were reverse transfected with siTOX or control siRNA SiCON at 16.5 nM. The former activates cellular death response while the latter does not target any known genes in the human genome. Total cell numbers at days 1, 2, 6, and 12 post transfection were compared between siTOX and siCON. SiTOX reduced cell number by 23% (day 1), 84% (day 2), 83% (day 6), and 72% (day 12) compared to siCON, without any noticeable cytotoxic effect in siCON treated cells (Additional file 4: Figure S4). Based on the above results, future experiments were conducted using 16.5 nM of siRNA to achieve 80–90% of transfection efficiency.
To examine the role of RGS2 and RGS4 in ad-hMSCs differentiation into adipocytes and osteocytes, siRNAs commercially validated against two different regions of the RGS4 mRNA (siRGS4-8 and siRGS4-10) and RGS2 mRNA (siRGS2-2 and siRGS2-3) were tested. SiRNA was transfected into cells at 2 days (D-2) prior to adipogenic differentiation initiation (D0 AIM). Expression of RGS2 and RGS4 in transfected ad-hMSCs were examined at day 1, 3, 5, 7, and 12 post adipogenic initiation with AIM containing 1.0 µM DEX or osteogenic initiation with OIM containing 0.2 µM DEX (Fig. 3). Expression level of RGS2 and RGS4 in each treatment group was normalized against the expression level of HSP90 and then graphed relative to its normalized expression in siCON control group at the same time point.
In samples treated with AIM, RGS4 mRNA was significantly (p < 0.05) lower at day 3 (39%), 5 (32%), and 7 (48%) in siRGS4-8 treatment groups compared to siCON controls (100%) (Fig. 3a, b). Similarly, in siRGS4-10 treatment groups, RGS4 mRNA was also significantly (p < 0.05) lower at day 3 (56%), 5 (36%), 7 (31%) and 12 (65%) compared to controls (100%) (Fig. 3a, b). In samples treated with OIM, expression of RGS4 was significantly (p < 0.05) lower in siRGS4-8 samples at day 1 (51%), 3 (39%), 7 (41%) and 12 (78%) compared to siCON (100%) (Fig. 3c, d). Likewise, in siRGS4-10 treatments, RGS4 mRNA was significantly (p < 0.05) lower at day 1 (65%), and 3 (23%), 7 (57%) and 14 (76%) (Fig. 3c, d). Overall, siRGS4 resulted in around 50–75% expression knockdown in RGS4 expression at the RNA level and there was no significant difference between siRGS4-8 and siRGS4-10 in RGS4 expression knockdown at any time point tested during adipogenesis or osteogenesis.
To examine the expression knockdown of RGS4 at the protein level, western blots were carried out using two types of antibodies that recognize two different motifs of RGS4 protein separately (see “Methods”). One binds to the C-terminal sequence (amino acids 182–205) outside of the RGS domain (amino acids 62–178) and detects the RGS4 isoform 3 product at 34 kDa. The other binds to the N-terminal sequences (amino acids 40–82) and detects the RGS4 isoforms 1 and 2 both at about 23 kDa. Expression level of RGS4 was compared between siRGS4-8 and siRGS4-10 transfected cells and siCON transfected cells at day 1, 2, 3, 4, 5 and 7 post AIM or OIM treatment initiation. Expression level remained similar among all groups at all time points examined (data not shown), except for day 7, when expression of RGS4 isoform 3 was consistently, thought slightly, down regulated in siRGS4-8 and siRGS4-10 transfected cells as compared to in siCON transfected cells by about 30 and 20% in AIM and OIM treatment condition, respectively (expression in OIM is shown in Additional file 5: Figure S5A). This delayed and subtle change at the protein level may imply much greater RGS4 protein stability as compared to its RNA transcript, in addition to other plausible causes (see “Discussion”).
In samples treated with AIM, RGS2 mRNA was significantly (p < 0.05) lower in siRGS2-2 samples at day 1 (84%), 3 (73%), 5 (83%), and 7 (80%) compared to siCON (100%) (Fig. 3e, f). The effect of siRGS2-2 was gone by day 12. Similarly, in siRGS2-3 treatments, RGS2 mRNA was significantly (p < 0.01) lower at day 1 (71%), and 3 (56%), 5 (63%), 7 (68%) and 12 (79%) (Fig. 3e, f). In samples treated with OIM, RGS2 mRNA was significantly (p < 0.05) lower in siRGS2-2 samples at day 1 (73%), and non-significantly lower by day 3 (86%), 7 (80%), and 12 (83%) (Fig. 3g, h). In siRGS2-3 samples, RGS2 mRNA was significantly (p < 0.05) down regulated at day 1 (51%), day 3 (63%), day 7 (75%), but resumed to control level by day 12 (103%) (Fig. 3g, h). Overall, siRGS2-2 and siRGS2-3 resulted in expression knockdown of RGS2 by 20–30 and 30–50%, respectively.
Since upregulation of RGS2 expression upon adipogenic initiation was significantly greater in Hyclone CM based AIM as compared to HI-FBS CM based AIM, and considering that the expression knockdown by siRGS2 was modest in Hyclone CM based AIM (by 20–50%), we wondered whether siRGS2 would have greater knockdown in HI-FBS CM based AIM due to lower basal level of RGS2 expression, and hence greater phenotypic effect. Similar to previous studies, expression knockdown of RGS2 by siRGS2-2 and siRGS2-3 was evaluated at days 1, 3, 5, 7, and 14 post treatment initiation with HI-FBS CM based AIM at 48 h after siRNA transfection. In siRGS2-2 samples, expression of RGS2 was significantly lower (p < 0.05) at day 3 (69%), 5 (60%) and 7 (51%), but resumed to control levels (97%) at day 12 as compared to siCON samples (100%) (Additional file 6: Figure S6). Similarly, in siRGS2-3 samples, expression of RGS2 was significantly lower (p < 0.05) at day 1 (43%), 3 (34%), 5 (32%), and 7 (49%) and resumed to control level by day 12 (94%) compared to siCON (100%) (Additional file 6: Figure S6).
Expression knockdown of RGS2 at the protein level was also examined by western blots in siRGS2-2, siRGS2-3 or siCON treated cells under AIM and OIM treatment conditions using Hyclone CM based media. Consistently, expression of RGS2 was down regulated by 30–40% in siRGS2-2 cells and 60–70% in siRGS2-3 cells as compared to in siCON cells on day 2 post AIM or OIM treatment initiation (expression in OIM is shown in Additional file 5: Figure S5B), which correlates well to the level of expression knockdown detected at the RNA level as shown above.
In conclusion, during early adipogenic and osteogenic treatments in Hyclone CM based media, both siRGS4-8 and siRGS4-10 downregulated RGS4 expression by about 50–75% at the RNA level, but only about 20–30% expression knockdown was detected in isoform 3 of RGS4 at the protein level, whereas siRGS2-2 and siRGS2-3 downregulated RGS2 expression by 20–30 and 30–50% respectively at the RNA level and similarly by 30–40 and 60–70% respectively at the protein level. Additionally, in HI FBS CM based AIM condition, siRGS2-3 exerted a greater level of gene silencing (by 50–70%) compared to siRGS2-2 (by 30–50%) at the RNA level, both of which are greater than their respective silencing effect in Hyclone CM based AIM. Next, we examined the effect of expression knockdown induced by siRGS2 and siRGS4 on adipogenic and osteogenic differentiation of hMSCs.
Expression knockdown of RGS2 and RGS4 exerts different levels of inhibitory effect on adipogenic differentiation of ad-hMSCs
To investigate the role of RGS2 and RGS4 in adipogenesis, we measured the effect of their expression knockdown induced by siRNA on several metrics of adipogenesis. As described previously, ad-hMSCs were reverse transfected with 16.5 nM of control (siCON) or targeted siRNA in Hyclone growth media (CM). After 48 h, adipogenesis was initiated by AIM with 1.0 μM DEX. After 12 days of AIM treatment, with media change at 48-h intervals, cells were fixed and stained with DAPI (nuclear stain) and OilRedO (oil droplet staining). Overlapping images of DAPI and OilRedO stained cells were taken from multiple wells of each treatment group for total cell counting, adipocytes counting and area measurements of stained lipid droplets in OilRedO images, and OilRedO dye was subsequently extracted with isopropanol and quantified by absorbance reading at 515 nm (see “Methods”).
For RGS4 expression knockdown, whole-well images showed noticeably lower intensity of OilRedO stains in siRGS4-10 but not siRGS4-8 groups compared to siCON control (Fig. 4a). Correspondingly, OilRedO quantification was significantly lower in siRGS4-10 treatment group (82%, p < 0.05) compared to siCON controls (100%), and the difference between siRGS4-8 treatment group (98%) and siCON control (100%) was insignificant (Fig. 4b). Consistently, area measurements of stained oil droplets were significantly lower in siRGS4-10 treatment groups (42%, p < 0.01) compared to siCON (100%), but insignificantly lower in RGS4-8 (85%) compared to siCON (100%) (Fig. 4c, d). Differences in total fat accumulation could be a result of variation in adipocyte numbers and/or variation in lipid accumulation within individual adipocytes. To determine the cause, total cell counts and adipocyte cell counts were determined based on DAPI nuclear stain and manual identification of mature adipocytes in OiRedO images respectively. Total cell numbers were significantly lower in both siRGS4-8 (88%, p ≤ 0.05) and siRGS4-10 (87%, p ≤ 0.05) treatment groups compared to siCON controls (100%) (Fig. 4e). Adipocyte cell numbers were even more drastically lower in both siRGS4-8 (50%, p < 0.05) and RGS4-10 (17%, p < 0.01) treatment groups compared to siCON controls (100%) (Fig. 4f). Percentage of adipocytes calculated by adipocytes number/total cell number was also significantly lower in siRGS4-8 (57%, p < 0.01) and siRGS4-10 (21%, p < 0.01) treatment groups compared to siCON controls (100%) (Fig. 4f). Overall, expression knockdown of RGS4 by siRGS4 resulted in significantly decreased total fat accumulation, total cell numbers, total adipocyte numbers and differentiation efficiency as reflected by percentage of adipocytes, with siRGS4-10 exerting greater effect than siRGS4-8.
For RGS2 expression knockdown, there was no noticeable difference in OilRedO staining intensity (Fig. 5a). OilRedO quantification was not significantly different between siRGS2-2 (99%) or siRGS2-3 (91%) treatment groups and siCON controls (100%) neither (Fig. 5b). Consistently, total area (pi2) measurements of OilRedO stained oil droplets trended lower in both siRG2-2 (81%) and siRGS2-3 (80%) treatment groups compared to siCON controls (100%), but the difference was not statistically significant (Fig. 5c, d). Nuclear counts in siRGS2-2 (96%) and siRGS2-3 (97%) slightly but consistently trended lower than siCON controls (100%), though statistically insignificant neither (data not shown). Overall, siRGS2 did not significantly affect total fat areas or total cell numbers as compared to siCON treatment in Hyclone CM based AIM condition.
The effect of siRGS2 on adipogenesis induced by HI FBS CM based AIM was also analyzed. In contrary to Hyclone CM based AIM condition, OilRedO stain intensity in both siRGS2-2 and siRGS2-3 treatments appeared visually reduced compared to siCON (Fig. 6a). Total fat accumulation quantification by OilRedO dye extraction however was only significantly lower in siRGS2-2 (86%, p < 0.05) but not in siRGS2-3 (96%) treatment groups as compared to siCON controls (100%) (Fig. 6b). Consistently, total area measurements (pi2) of stained oil droplets was significantly lower in siRGS2-2 (55%, p < 0.05) and only trended lower in siRGS2-3 (80%, p < 0.1) treatments as compared to in siCON controls (100%) (Fig. 6c, d). Subsequently we determined whether decreased total fat accumulation was the result of reduction in adipocyte numbers and/or differentiation efficiency. Total nuclear counts were only significantly lower in siRGS2-2 (87%, p < 0.05) but not in siRGS2-3 (92%) treatment groups compared to siCON controls (100%) (Fig. 6e). Total adipocyte number trended lower in both siRGS2-2 (82%, p < 0.1) and siRGS2-3 (86%) treatments compared to siCON controls (100%) (Fig. 6f). Percent of adipocytes in siRGS2-2 (96%) and siRGS2-3 (90%) groups were not significantly different from siCON controls (100%) (Fig. 6f). Overall, in the HI FBS CM based AIM induced adipogenic differentiation, siRGS2-3 had mild inhibitory effect that was statistically deemed insignificant, but siRGS2-2 treatment significantly inhibited total fat accumulation as compared to siCON, which was likely the consequence of significantly reduced total number of cells, as differentiation efficiency determined by percentage of adipocytes was not significantly different between siRGS2-2 and siCON.
In conclusion, expression knockdown of RGS4 by 50–75% significantly inhibited adipogenic differentiation of hMSCs by reducing total adipocytes and adipogenic differentiation efficiency, with siRGS4-10 exerting greater effect than siRGS4-8. Expression knockdown of RGS2 also exhibited similar inhibitory effect in HI-FBS CM based AIM but not Hyclone CM based AIM conditions, likely due to greater expression knockdown in the former vs. the latter, with siRGS2-2 exerting greater effect than siRGS2-3. Such effect was at least partly due to reduced total adipocytes as the result of reduced total cell numbers, without affecting differentiation efficiency.
Effects of siRGS2 and siRGS4 on the expression of adipogenic markers
Expression knockdown of RGS4 by siRGS4 resulted in significantly decreased adipogenesis in part due to reduced total cell numbers. Since differentiation efficiency as reflected by percentage of adipocytes was also reduced, it indicated that decreased adipocyte number in siRGS4 treatment groups might not be solely due to reduction in total cell number and siRGS4 might affect adipogenesis directly, resulting in decreased differentiation efficiency. On the other hand, siRGS2 had insignificant effect on adipogenesis in response to Hyclone CM based AIM but significant inhibitory effect on adipogenesis in response to HI FBS CM based AIM, without significantly affecting adipogenic differentiation efficiency. This indicated that siRGS2 did not likely affect adipogenesis directly. To test the above, the effect of siRGS2 or siRGS4 on the expression of selected adipogenic makers, PPARγ, C/EBPα and LPL, was measured at day 1, 3, 5, 7, and 12 post adipogenic induction (Hyclone CM based AIM) in hMSCs that had been subjected to siRGS2/siRGS4 or siCON transfection. Similar to previous expression analyses, expression of those genes in siRGS2/siRGS4 treated cells was measured by RT-PCR and compared to its value in siCON treated samples at the same time point, after normalization to the expression level of internal control HSP90.
In siRGS4 samples, expression levels of PPARγ and C/EBPα in siRGS4-8 were not significantly different from siCON controls at all time points examined, except for PPARγ upregulation at day 1 (130%, p < 0.05) and C/EBPα downregulation at day 12 (20%, p < 0.05) compared to siCON (100%) (Fig. 7a, b). However, in siRGS4-10 samples PPARγ expression level overall trended lower while C/EBPα was significantly down regulated at all time points (15–25%, p < 0.05) compared to siCON controls (100%) (Fig. 7a, b). Expression of LPL on the other hand was significantly down regulated in both siRGS4-8 and siRGS4-10, with 45% at day 5, 77% at day 7 and 23% at day 12 in the former and 18% at day 3, 33% at day 5, 22% at day 7 and 6% at day 12 in the latter samples as compared to siCON (100%) (Fig. 7c). Overall, expression of both C/EBPα and LPL were significantly down regulated by siRGS4-10 at multiple time points during adipogenic differentiation, whereas only LPL was down regulated by siRGS4-8 at multiple time points, which is consistent with the more disruptive effect of siRGS4-10 on adipogenic differentiation of hMSCs as compared to siRGS4-8.
In siRGS2 samples, expression level of PPARγ was slightly but significantly lower in siRGS2-2 treatments (84–90%) as compared to siCON (100%) on day 5, 7 and 12, and similar difference between siRGS2-3 and siCON was observed on day 1 and day 12 (Fig. 7e). Expression of C/EBPα was also slightly but significantly downregulated by siRGS2-2 (89%) and siRGS2-3 (77%) on day 3 compared to siCON (100%), but remained unchanged at the other time points (Fig. 7f). Expression of LPL was upregulated in siRGS2-2 samples at day 12 (129%, p < 0.05) compared to siCON controls (100%), but was not significantly changed at the other time points. In siRGS2-3 samples, levels of LPL were slightly but significantly lower at day 3 (83%) and 5 (72%) compared to siCON samples (100%), but remained insignificantly different at the other time points (Fig. 7g). Overall, both siRGS2-2 and siRGS2-3 had a subtle suppressive effect on the expression of both PPARγ and C/EBPα, and only siRGS2-3 appeared to have a subtle suppressive effect on the expression of LPL, consistent with the overall mild and insignificant effect of siRGS2 on total fat accumulation in Hyclone CM based AIM condition.
Since siRGS2 exerted significant inhibitory effect on adipogenic differentiation of hMSCs induced by HI-FBS CM based AIM, expression of all four adipogenic marker genes was also examined in such condition. Expression of PPARγ was slightly but significantly down regulated by siRGS2-2 at day 3 (76%, p < 0.05) and day 5 (88%, p < 0.05) but not by siRGS2-3 as compared to siCON (100%) (Fig. 8a). C/EBPα expression on the other hand was upregulated in siRGS2-2 treatment groups at day 7 (120%, p < 0.05) and 12 (160%, p < 0.05), but slightly downregulated in siRGS2-3 at day 3 (73%, p < 0.05) compared to siCON (100%) (Fig. 8b). LPL expression was only downregulated by siRGS2-2 (50%, p < 0.05) at day 5 but not by siRGS2-3 compared to siCON (100%) (Fig. 8c). Overall, siRGS2-2 slightly down regulated expression of PPARγ and LPL, but upregulated C/EBPα, whereas siRGS2-3 had minimum effect on the expression of these genes except for transient downregulation of C/EBPα. This is consistent with previous observation that compared to siRGS2-3, siRGS2-2 exerted greater inhibitory effect on adipogenesis induced by HI FBS CM based AIM. In addition, effect of siRGS2-2 on adipogenic gene expression was only slight, consistent with previous observation that siRGS2-2 did not significantly affect differentiation efficiency determined by percentage of adipocytes, and its inhibitory effect on adipogenesis was mainly likely due to reduced adipocytes as the result of reduced total cell numbers and possibly reduced fat accumulation per adipocyte as well.
In conclusion, consistent with their different levels of inhibitory effect on the adipogenic outcome of hMSCs, siRGS4 exerted significantly greater level of inhibition on the expression of adipogenic marker genes (PPARγ, C/EBPα, and LPL) than siRGS2. In addition, siRGS4-10 downregulated all three genes whereas siRGS4-8 only inhibited LPL, which is also consistent with the more disruptive effect of siRGS4-10 on adipogenic differentiation of hMSCs as compared to siRGS4-8.
Effect of siRGS2 and siRGS4 in osteogenic differentiation of hMSCs
To investigate the potential role of RGS2 and RGS4 during osteogenic differentiation of hMSCs, we applied the same D-2/D0 siRNA transfection approach. Briefly, even number of ad-hMSCs were reverse transfected with xtremeGENE/siRNA complex at 16.5 nM in Hyclone control (CM) media. After 48 h, osteogenesis was induced by 0.2 μM DEX OIM media, which was subsequently changed every 48 h. After 18–26 days of OIM media treatment, cells were fixed and stained with alizarin red S, which specifically stains for calcific deposit (hydroxylapatite) by osteocytes. Alizarin Red S dye was subsequently extracted with acetic acid and quantified calorimetrically at 405 nm as a measurement of osteogenic differentiation efficiency (see “Methods”).
In comparison to siCON treatments, there was increased as well as earlier onset of calcific deposit (day 11) present in siRGS4-10 treatment groups but not so much in siRGS4-8 groups (Fig. 9a, top row), which could also be visually confirmed by increased amount of Alizarin Red S stain in siRGS4-10 treatment groups at the end of differentiation (day 18–24) (Fig. 9a, middle row). On the other hand, mineral deposit was decreased in both siRGS2-2 and siRGS2-3 samples compared to siCON groups (Fig. 9a, bottom row). Consistently, Alizarin Red S quantification was significantly higher in siRGS4-10 treated samples (169%, p < 0.05) (Fig. 9b), but significantly lower in both siRGS2-2 (84%, p < 0.05) and siRGS2-3 (68%, p < 0.05) treated samples compared to siCON controls (100%) (Fig. 9c). Nuclear count revealed no significant difference between siCON and any siRGS4 or siRGS2 treatment groups (data not shown).
In conclusion, siRGS4 and siRGS2 had opposing effect on osteogenic differentiation of hMSCs, with the former promoting while the latter inhibiting the process, without affecting total cell numbers. In addition, similar to a more disruptive effect of siRGS4-10 on adipogenic differentiation of hMSCs as compared to siRGS4-8, the former also demonstrated a greater enhancing effect on osteogenic differentiation of hMSCs compared to the latter.
Effects of siRGS2 and siRGS4 on the expression of osteogenic markers
Since siRGS4 promoted osteogenic differentiation of hMSCs while siRGS2 inhibited it without affecting total cell numbers, it implied a direct effect on osteogenic differentiation. Effect of siRGS4 and siRGS2 on the temporal expression of known osteogenic markers, Runx2, Osteocalcin (OC) and alkaline phosphatase (ALP) was further evaluated. Runx2 is an osteogenic master regulator [14]. OC encodes a bone specific protein synthesized by osteoblast and serves as a marker of osteogenic maturation [59], while ALP encodes an enzyme that function to promote mineralization by increasing phosphate concentrations [60, 61]. Since type II/p57 isoform of Runx2 has been shown to be bone specific [62], primers amplifying specifically the N-terminal region of the gene that encodes the bone-specific MASNS polypeptide domain was used in analyzing the expression of Runx2.
In siRGS4 OIM treatments, Runx2 expression was upregulated in both siRGS4-8 and siRGS4-10 treated cells as compared to siCON controls starting on D3 post OIM treatment initiation, but the enhancement is clearly much stronger in siRGS4-10 samples, at about 2- to 4-fold higher level than in siRGS4-8 (Fig. 10a), which is consistent with the greater effect of siRGS4-10 on promoting osteogenic differentiation. For expression of OC, no significant difference between siCON and siRGS4-8 or siRGS4-10 samples was observed at any time point analyzed (Fig. 10b). Expression of ALP was also very similar between siRGS4-8/siRGS4-10 and siCON at all time points, except for day 7, when it was slightly down regulated in siRGS4-8 (84%, p < 0.05) (Fig. 10c). Overall, Runx2 was upregulated by both siRGS4-8 and siRGS4-10, but at much greater level by the latter. ALP appeared to be transiently downregulated by siRGS4-8 but remained unaffected by siRGS4-10.
In siRGS2 OIM treatments, Runx2 was down regulated by both siRGS2-2 and siRGS2-3 treatment starting at day 1 and lasted at least until day 7, at about 52–88% expression level of its normal expression (p < 0.05) in siCON (100%) (Fig. 10d). Expression of OC in siRGS2-2 and siRGS2-3 did not significantly differ from siCON treatment at day 1, but was transiently upregulated at day 3 (146%, p < 0.05) and day 5 (120%, p < 0.05) in siRGS2-2 and at day 14 (124%, p < 0.05) in siRGS2-3 compared to siCON (100%) (Fig. 10e). Finally, expression of ALP was slightly downregulated in siRGS2-2 (84%) and siRGS2-3 (87%) but then was transiently upregulated at day 3 (124%, p < 0.05) and day 5 (122%, p < 0.05) in siRGS2-2 and at day 14 (127%, p < 0.05) in siRGS2-3 compared to siCON (100%) (Fig. 10f). Overall, Runx2 was slightly downregulated by both siRGS2-2 and siRGS2-3 from day 1 until at least day 7. ALP expression was also detected to be slightly downregulated on day 1, but was shifted to slight upregulation on later days. Expression of OC was also slightly upregulated by siRGS2-2 or siRGS2-3 at day 3 and 7 or day 14, respectively, following the same trend as ALP expression on those days.
In conclusion, Runx2 was upregulated by both siRGS4-8 and siRGS4-10 throughout osteogenic differentiation but downregulated by siRGS2-2 and siRGS2-3. RGS4 silencing had no significant effect on the expression of OC or ALP, while RGS2 silencing transiently downregulated ALP expression early on before upregulating it along with OC at later time points.