2.1. Differentiation and mineralization of BMSCs in SMG
Exposure to SMG does not affect BMSC viability, either at 8 (T8) or 28 days (T28) (Fig. S1). The expression of osteogenic gene markers was assessed by qRT-PCR. Runt related transcription factor 2 (Runx2), alkaline phosphatase tissue non-specific (Alpl), decorin (Dcn), and collagen type I (Col1a1) were analysed as early markers, and collagen type III (Col3a1), bone morphogenetic protein 2 (Bmp2), and bone sialoprotein 2 (Ibsp), used as late markers. Runx2, Dcn and Col1a1 were not significantly affected by RPM exposure (data not shown), while Col3a1, Alpl, Ibsp and Bmp2 expression was downregulated by microgravity exposure at both T8 and T28 (Fig. 1A).
Differentiation was also evaluated at the protein level, by immunostaining of COL1A1 and the osteocalcin (BGLAP, also known as bone gamma carboxyglutamate protein) at 28 days (Fig.1B) and by immunoenzymatic quantification of the bone ECM protein deposition at both time points (T8 and T28) (Table 1). Immunofluorescence analysis of both proteins, COL1A1 (Fig.1B, left side) and BGLAP (Fig.1B, right side), showed a significant reduction of fluorescence intensity and protein area in samples exposed to RPM compared to ground control (GC).
Immunoenzymatic quantification was done on COL1A1, COL3A1, DCN, fibronectin (FN), BGLAP, osteonectin (SPARC), and osteopontin (SPP1, also known as secreted phosphoprotein 1) (Table 1). The only statistically significant variations were seen in the levels of COL1A1 and SPP1. In detail, COL1A1 was found upregulated at 28 days with respect to 8 days, both in GC and RPM conditions (Table 1, GC - T8 vs T28 and RPM – T8 vs T28), indicating osteogenic differentiation was undergoing in both conditions. Although, at 28 days in RPM the COL1A1 content was lower than in GC condition (Table 1, T28 - GC vs RPM), suggesting a reduced osteogenic differentiation. SPP1 was found upregulated at 8 days in RPM with respect to 8 days in GC condition (Table 1, T8 - GC vs RPM). However, the SPP1 content at 28 days in RPM was lower than at 8 days in RPM (Table 1, RPM - T8 vs T28). The immunoenzymatic quantification of COL3A1, DCN, FN, BGLAP and SPARC showed non-significant variations among the classes. Taken together these results indicate lower or delayed differentiation of BMSCs when grown on the RPM.
Table 1
Extracellular matrix protein evaluation by indirect ELISA. Indirect enzyme-linked immunosorbent assay (ELISA) quantifying the amount of specific protein detected per μg of total protein content. Statistical significance was assessed via Student T-test, comparing the GC and RPM conditions at both time points. In the column p the p-value is reported. * = p<0.05.
Proteins (μg/μg total proteins)
|
T8
|
T28
|
GC
|
RPM
|
GC
|
RPM
|
GC vs RPM (p)
|
GC
|
RPM
|
GC vs RPM (p)
|
T8 vs T28 (p)
|
T8 vs T28 (p)
|
COL1A1
|
0.089 ± 0.004
|
0.091 ± 0.002
|
0.563
|
0.189 ± 0.019
|
0.123 ± 0.008
|
*0.045
|
*0.018
|
*0.030
|
COL3A1
|
0.390 ± 0.016
|
0.387 ± 0.013
|
0.850
|
0.409 ± 0.001
|
0.411 ± 0.006
|
0.684
|
0.243
|
0.142
|
DCN
|
0.261 ± 0.030
|
0.257 ± 0.010
|
0.879
|
0.258 ± 0.006
|
0.257 ± 0.021
|
0.961
|
0.898
|
0.994
|
FN
|
12.65 ± 0.213
|
13.12 ± 0.414
|
0.296
|
31.47 ± 27.76
|
20.82 ± 21.80
|
0.711
|
0.439
|
0.667
|
BGLAP
|
20.52 ± 2.095
|
23.58 ± 0.178
|
0.176
|
19.91 ± 0.943
|
20.10 ± 1.435
|
0.893
|
0.744
|
0.077
|
SPARC (x103)
|
8.308 ± 0.184
|
8.761 ± 1.095
|
0.623
|
8.449 ± 0.567
|
9.144 ± 0.223
|
0.248
|
0.771
|
0.675
|
SPP1
(x103)
|
114.4 ± 3.121
|
132.0 ± 1.112
|
*0.017
|
118.2 ± 0.646
|
118.6 ± 3.013
|
0.869
|
0.231
|
*0.027
|
ALPL enzymatic activity was significantly lower in SMG at four investigated time points over the 28 days (Fig. 2A) in agreement with ALPL transcript expression, downregulated in RPM at T8 and at T28 (Fig. 1A). Additionally, the mineralization of the ECM determined by measuring crystal size revealed a significant reduction in the dimension and an altered percentage distribution (Fig. 2B, C, D), suggesting impaired osteoblasts activity. Indeed, on RPM it was already possible to observe smaller crystals at T14 compared to GC conditions. Alizarin red positive spots were almost completely absent in RPM at T8 but they were present at T28, again indicating impaired osteoblasts activity. In accordance, alizarin red absorbance showed a significant reduction of calcium deposits at T8 and T28 in RPM samples (Fig. 2E). These results indicated a reduction in the osteogenic phenotype when BMSCs were exposed to SMG. However, the synthesis of some ECM proteins and the mineralization capacity of the differentiating BMSCs were not completely impaired.
2.2. Cytoskeleton investigation with bio-imaging in SMG
F-Actin and β-tubulin were investigated on BMSCs during osteogenic differentiation in order to determine the long-term effect of SMG on cytoskeleton proteins by immunofluorescence (Fig. 3A). Intensity analysis of the cells revealed that during the first 24 h of microgravity there was a significant decrease of both tubulin and actin mean intensity; actin filaments (Fig. 3B-1) and the tubulin network (Fig. 3B-2) showed a recovery after 4 days. Indeed, after 28 days the percentage of tubulin area in cells exposed to SMG was not significantly different compared to GC (Fig. 3B-3). Deeper analysis, initially performed with “fire lookup table” (Fig. 3C-3 and 4) and later with mean intensity and fraction area measurements per belt, revealed that distribution of β-tubulin within cells was still altered after 28 days. In particular, the mean intensity of samples exposed to SMG was significantly higher in proximity to the centrosome and the surface occupied by tubulin was lower in the cell periphery (Fig. 3C-5 and 6; Fig. 3D-1 and 2). This evidence shows that a cytoskeletal adjustment of β-tubulin and F-actin fibres occured during the first two days of SMG exposure. However, these changes were almost completely reversed at 28 days, leaving the cells with a reduced β-tubulin fluorescent area in their more peripheral cytosolic portion.
2.3 Proteomics investigation of the protein groups affected by SMG
To understand proteome variations caused by SMG during the osteogenic differentiation of BMSCs, we applied a proteomics approach based on mass spectrometry label-free quantification (Fig. 4A). The proteomics investigation allowed the identification of 4312 protein groups (PGs) in total (Table S1). Following data pre-processing (ratio on time zero, T0, filtering for valid values, log2 transformation, and missing data imputation; Fig. S2) the number of PGs was reduced to 4114. To narrow down the number of PGs relevant for this study, multiple sample tests were used to statistically infer the differentially abundant PGs (DAPGs). To simplify the statistical model, and following data interpretation, the two independent variables present in the experiment (time and SMG exposure) were fused into one unique independent variable. The classes of the unique independent variable were therefore four: T8_GC (BMSCs in osteogenic differentiation for 8 days under gravity control), T8_RPM (BMSCs in osteogenic differentiation for 8 days under SMG), T28_GC (BMSCs in osteogenic differentiation for 28 days under gravity control) and T28_RPM (BMSCs in osteogenic differentiation for 28 days under SMG). 486 PGs, reduced to 481 following an isoforms check, were differentially abundant among the four classes, and post-hoc THSD was applied to annotate class-specific variation for each DAPG (Table S2).
Principal component analysis (PCA) of the sample replicates showed that 59% of variance was due to first two components (Fig. 4B). The replicates were clustered by the PCA into five groups, based on the two independent variables (time and SMG exposure). T8 groups appeared more distant from T28 groups than GC from RPM, suggesting that time was generating more variance than SMG on BMSCs proteome.
GO annotation overrepresentation analysis (ORA) of the 481 DAPGs showed that 13 cellular component categories (GO-CC) were significantly enriched (Fig. S3). Interestingly, 49 DAPGs were mapped to the GO-CC ECM (p = 2.7x10-11), while 28 were mapped to the mitochondrial matrix (p = 3.1x10-3), 47 to the cell-substrate junction (p = 3.8x10-13), and 30 to the actin cytoskeleton (p = 1.5x10-3).
Z-scored values of the top 10 PGs most downregulated (ABCC3, ABCC4, ACSL1, ATG9A, ATP1A1, ATP2B1, EIF4A2, MT-ATP6, SLC44A1, TMEM63A) and the top 10 PGs most upregulated (ABCF3, CRAT, FKBP10, MTHFD1L, NSUN2, NT5DC3, PUS7, QARS, SELENON, XIRP1) in RPM were hierarchically clustered (Fig. 4C). Interestingly, many of these 20 DAPGs are under the control of stemness master regulators, such as POU5F1 and NANOG.
To understand how the SMG exposure was affecting PGs regulation over time, the 481 DAPGs were divided in a pie chart (Fig. 4D) based on their regulation in SMG at 8 and 28 days. Of those 481 DAPGs, 15.2% (73 PGs) and 6.6% (32 PGs) were respectively down and upregulated exclusively at T8, against the 3.5% (17 PGs) and 5.8% (28 PGs) that were, respectively, down and upregulated solely at T28. An additional 5.8% (28 PGs) and 3.3% (16 PGs) were found down and upregulated at both time points. The remaining 59.8% of DAPGs were attributed to the BMSCs maturation during time, comparing the conditions T8_GC vs T28_GC and T8_RPM with T28_RPM. This distribution highlighted the fact that a reduction in the overall metabolism of the BMSCs underwent at early time points (8 days) and was relieved at the 28th day. An increasing upregulation was instead present in these cells.
The enrichment analysis resulted in overrepresentation of 178 unique GO-BP terms (Table S3) and helped to narrow down the cellular processes mainly affected by SMG. These 178 GO-BPs were represented in 4 charts based on their down and upregulation data, at 8 and 28 days (Fig. S4-S7). They presented redundant DAPGs in their list, but this also ensured that the biological significance of the proteins was not underestimated (as one protein can be, and usually is, involved in multiple pathways).
In general, the overall proteomics coverage was high with respect to previous works (4312 identified PGs). However, the GO-CCs annotation of the DAPGs highlighted a polarized enrichment of non-nuclear proteins. Results from the 481 DAPGs suggest that major changes occured at early time points (8 days), with the 15.2% of all DAPGs being downregulated by SMG at 8 days.
2.4 Functional annotation of the DAPGs and relative enriched GO-BPs found in SMG
To rationalize the GO-BPs revealed by enrichment analysis, we clustered them into 2 macro categories, each comprising 5 categories which were functionally correlated. The cell fate macro category included proliferation, differentiation, adhesion, signalling, and death (Fig. 5). The cell metabolism macro category ,oth the other hand, was divided in carbohydrates, lipids, proteins, and nucleic acids metabolisms, and transport (Fig. 5). Proteomics data showed the enrichment of 13 GO-BPs related to BMSCs and osteoblasts proliferation. Among the enriching proteins, a significant reduction at 8 days in the expression of 7 DAPGs (COL8A1, CSPG4, DYNC1H1, MTOR, NCSTN, PLPP3, and PRKDC) was followed by a significant upregulation at 28 days of 6 DAPGs (CCN1, FBLN5, NFKB2, NRP2, NSUN2, and SEMA3C) (Fig. 5 - Proliferation). Notice that CCN1 and FBLN5 were annotated to the GO-BP “positive regulation of osteoblast proliferation” (GO:0033690). MTOR and PRKDC can also be associated with the PI3K/AKT/mTOR pathway.
16 out of 27 DAPGs under the differentiation category (Fig. 5 - Differentiation) were downregulated in RPM at 8 days (ATP1A1, ATP2B1, ATP2B4, CD47, COL11A1, COL3A1, COL6A3, FASN, HSPG2, ITGA1, ITGA11, MRC2, PTPN11, TMEM119, TNC, and VCAN), and only 6 of these were downregulated at 28 days (ATP1A1, ATP2B1, CD47, FASN, TMEM119, VCAN). All of these are annotated under or related to the GO-BP terms “osteoblast differentiation, ossification, skeletal system morphogenesis and bone development”. The other 11 DAPGs were found upregulated in RPM: 6 were upregulated at the earliest time point (ABR, ACTA1, LMNA, NSUN2, PAPSS1, PUS7) and 10 at the last time point (ABR, ACTA1, CCN1, FBLN5, LOXL1, NSUN2, PAPSS1, PUS7, SEMA3C, TGM2). Interestingly, MAP2K1 (also known as MAPKK 1, MKK1, ERK activator kinase 1, MAPK/ERK kinase 1, or MEK1) was found downregulated at 28 days with respect to 8 days, both in RPM and GC (Table S2). MAP2K1 can inhibit PPAR𝛾 activity in the nucleus and repress adipogenic differentiation. A non-significant increase of MAP1K2 in RPM with respect to GC might suggest a mild reduction adipogenesis (Table S2).
In the adhesion category (Fig. 5 - Adhesion) there were 30 enriched GO-BPs. Among them, 14 were downregulated in RPM at 8 days (ATP2A2, CD151, CD47, COL12A1, COL3A1, COL8A1, CSPG4, FLNA, FLNB, ITGA1, ITGA11, LRP1, MYADM, PLEC, PLPP3, PTPN11, TLN1, TLN2, TNC, VCAN) and 5 of these were also downregulated at 28 days (CD151, CD47, COL8A1, MYADM, VCAN), indicating a reduction in overall cell adhesion and migration properties. Interestingly, the downregulated DAPGs enriched for the integrin-mediated signalling pathway (GO:0007229 by CD47, ITGA1, ITGA11, PLPP3, and PTPN11), hemidesmosome assembly (GO:0031581 by CD151 and PLEC), cytoskeleton organization (GO:0007010 by FLNB, TLN1, and TLN2) and ECM organization (GO:0030198 by COL8A1, TCN, and VCAN), suggest a reduction in BMSC adhesion and an overall reorganization of the cytoskeletal fibres at the early time point (8 days). ABR, SORBS1, and ZYX were upregulated at 8 days in RPM and 7 were upregulated at 28 days (ALCAM, CCN1, FBLN1, FBLN5, ICAM1, NRP2, SEMA3C). The early (8 days) upregulation of SORBS1 and ZYX, enriching for stress fibre assembly, was in line with results derived from F-actin bioimaging analysis. Indeed, following a sudden reduction of stress fibres (shown as reduction in the mean fluorescent intensity) at T1h (Fig. 3A), there was a reorganization and reformation of those fibres during the following days (fluorescent area and intensity increased already at day 4 and remained comparable to GC condition) (Fig. 3A-B). The later upregulation of DAPGs involved in cell migration (ALCAM, NRP2, SEMA3C) suggests BMSCs started migrating again once they reorganized the cytoskeleton and adapted to SMG.
In the signalling category (Fig. 5 - Signaling) a total of 14 DAPGs were found, of which 8 were downregulated at 8 days (ABCC4, ATP2A2, CD47, COL3A1, MYADM, TNC, VCAN, WNK1) and 5 remained downregulated at 28 days. While 2 were upregulated at 8 days (ABR and MSN) and 4 were upregulated at 28 days in RPM (ABR, LOXL1, NRP2, PDK1). Among the proteins enriching the signalling category, 8 were also found to enrich the adhesion category (Fig. 5 – Adhesion) (ABR, ATP2A2, CD47, COL3A3, MYADM, NRP2, TNC, VCAN). This was possibly due to the fact that a reduction in cell adhesion and cytoskeletal reorganization events was the stimulus and/or response to the microgravity signal.
6 DAPGs enriched the cell death category (Fig. 5). Among them MTOR was downregulated at 8 days, ENDOG and PAK2 were upregulated at 8 days and CCN1, TIA1 and TIMP3 were upregulated at 28 days, indicating an overall upregulation of apoptosis. This is in line with the data on cell viable numbers, as we registered an almost constant number of alive BMSCs, due to a certain percentage of proliferation and a certain percentage of apoptosis.
Regarding the cell metabolism macro category (Fig. 5), we grouped the DAPGs involved in the metabolism of the different macromolecules. Carbohydrate metabolism (Fig. 5 – Carbohydrate) included simple and complex carbohydrates metabolic processes. In this category, CSPG4, DSE and VCAN were downregulated at 8 days, while SCARB2, SLC2A1 and VCAN were downregulated at 28 days. These proteins mostly enriched the biosynthetic pathways of lactose, chondroitin and dermatan sulfate biosynthesis (the last two being glycosaminoglycans - GAGs). PKM and SLC25A12 were upregulated at 8 days and PGM3 only at 28 days, which enriched glycolysis related GO-BPs. Interestingly, PKM and SLC2A1 were also involved in the aerobic glycolysis pathway. PDK1 is upregulated at 28 days in RPM with respect to GC, and PDK3 and PC are upregulated at 28 days with respect to 8 days, in both RPM and GC (Table S2). PFKL and PFKP were downregulated in both conditions at 28 days with respect to 8 days (Table S2).
Among the 11 DAPGs enriching lipid-related metabolic processes (Fig. 5 – Lipids), 8 were downregulated at 8 days (ACSL1, ALOX15B, ATP1A1, CEPT1, FASN, LRP1, SLC44A1, SLC44A2) and 6 at 28 days (ACSL1, ALOX15B, ATP1A1, CEPT1, FASN, SLC44A1). These downregulated proteins generally enriched lipid or lipidated molecules biosynthesis related pathways. Only ß-oxidation related proteins were found upregulated (ACOX1 and CRAT at 8 days and ACOX3 and CRAT at 28 days) and the leptin receptor (LEPR) was found upregulated at T28 in RPM with respect to T8 in RPM (Table S2), suggesting an increase in the basal metabolism undergoing in RPM at 28 days. Furthermore, ACADM, which is the catalyst for the first step of mitochondrial fatty acid beta-oxidation, was upregulated at 28 days in both conditions, while ACAA2, the catalyst for the last step of the mitochondrial beta-oxidation pathway, was downregulated at 28 days with respect to 8 days in both conditions (Table S2). So, overall lipid-related proteins suggest a general downregulation of lipid biosynthesis and upregulation of lipid degradation by oxidation, which is in line with a general spike of the basal metabolism.
In the protein metabolism – related pathways (Fig. 5 - Proteins) we found a total of 35 DAPGs. The DAPGs involved in protein N-glycosylation (DAD1, OSTC, STT3A, TUSC3), regulation of translational initiation (EIF4A2, MTOR, NCBP1) and other protein modification processes were downregulated in RPM, while DAPGs involved in the elastic fiber formation processes were upregulated (FBLN1, LOX1, LOX4, TIMP3). The synthesis of biglycan (BGN), bone-related proteoglycan, is significantly increased in RPM at 28 days (Table S2).
In the transport related processes (Fig. 5 - Transport), we annotated 26 different DAPGs and 22 of these were downregulated against the 4 DAPGs upregulated by SMG (ABR, EHD4, KRT18, NSF). 10 of the downregulated DAPGs were annotated to pathways of export from the cells and 4 are involved in calcium ion transport (ATP1A1, ATP2B1, ATP2B4, ATP2C1).
Regarding the nucleic acids metabolic processes (Fig. 5 – Nucleic acids), we found several DAPGs involved in RNA modification upregulated (NSUN2, QARS, SNRPE, SSB, WARS) in RPM, while DNA metabolic processes were mostly linked to DAPGs downregulated by RPM (CAD, MTOR, PRKDC). In contrast to the general tendency of having more downregulated proteins in RPM, in this category composed of 12 DAPGs, only 5 were downregulated and 7 upregulated.