GSK690693

ITGB1 promotes the chondrogenic differentiation of human adipose‑derived mesenchymal stem cells by activating the ERK signaling

Simin Luo1 · Qiping Shi2 · Wuji Li1 · Wenrui Wu1 · Zhengang Zha1

Received: 11 February 2020 / Accepted: 8 October 2020
© Springer Nature B.V. 2020

Abstract

Adipose-derived mesenchymal stem cell (ADSC) with a high capacity of chondrogenic differentiation was a promising candidate for cartilage defect treatment. This study’s objective is to study the roles of integrin β1 (ITGB1) in regulating ADSC chondrogenic differentiations as well as the underlying mechanisms. The identity of ADSC was confirmed by flow cytometry. ITGB1 gene was overexpressed in human ADSC (hADSC) by transfection with LV003-recombinant plasmids. Gene mRNA and protein levels were examined using quantitative RT-PCR and western blotting, respectively. Differen- tially expressed mRNAs and proteins were characterized by next-generation RNA sequencing and label-free quantitative proteomics, respectively. ERK signaling and AKT signaling in hADSCs were inhibited by treating with SCH772984 and GSK690693, respectively. ITGB1 gene overexpression substantially increased collagen type II alpha 1 chain (COL2A1), aggrecan (ACAN), and SRY-box transcription factor 9 (SOX9) expression but suppressed collagen type I alpha 1 chain (COL1A1) expression in hADSCs. Next-generation RNA sequencing identified a total of 246 genes differentially expressed in hADSCs by ITGB1 overexpression, such as 183 upregulated and 63 downregulated genes. Label-free proteomics charac- terized 34 proteins differentially expressed in ITGB1-overexpressing hADSCs. Differentially expressed genes and proteins were enriched by different biological processes such as cell adhesion and differentiation and numerous signaling pathways such as the ERK signaling pathway. ERK inhibitor treatment caused substantially enhanced chondrogenic differentiation in ITGB1-overexpressing hADSCs. ITGB1 promoted the chondrogenic differentiation of human ADSCs via the activation of the ERK signaling pathway.

Keywords ITGB1 · Mesenchymal stem cell · hADSC · Chondrogenic differentiation · ERK signaling · Cartilage effect

Introduction

Articular cartilages on diarthrodial joint surface have impor- tant junction-protecting roles by functioning as low-friction interface for motion and cushioning external impacts (Deng et al. 2016). Cartilage defects, due to trauma, inflammation,aging, and also genetic factors, are common intractable orthopedic disorders, featured by incapacity of self-repair because of poor blood supply in articular cartilage tissues, which could further progress into more severe lesions and even osteoarthritis (OA) without proper treatments (Deng et al. 2016; Nam et al. 2018). Nowadays, cartilage defects are usually treated by different surgical paradigms such as osteochondral autograft transfer, Pridie drilling, and abra- sion chondroplasty; however, they did not achieve sufficient recapitulation of native cartilage tissues (Deng et al. 2016).

The maintenance of cartilage functions greatly depends on chondrocytes that generate the key components of the extra- cellular matrix (ECM), such as collagen, proteoglycans, and hyaluronic acid (Nam et al. 2018). In the last two decades, stem cells with high capacity of chondrocyte regeneration have been a promising strategy for the treatment of cartilage defects (Deng et al. 2016; Hellingman et al. 2012; Nam et al. 2018; Pastides et al. 2013).

Mesenchymal stem cells (MSCs) have multipotent poten- tials to differentiate into different cell lineages, such as chon- drocytes, osteoblasts, myocytes, adipocytes, and even neural cells (Mathieu and Loboa 2012; Steward and Kelly 2016). Adipose-derived mesenchymal stem cells (ADSCs) are multipotent stromal cells isolated from the stromal vascular fraction of adipose tissues (Hamid et al. 2012; Stromps et al. 2014). The therapeutic potentials of ADSCs have gained great attentions among the stem cell and regeneration medi- cine research community in recent years, chiefly because of the high yield and less-invasive harvesting procedure during ADSC isolation in comparison with bone marrow-derived mesenchymal stem cells (BMSCs) (Nam et al. 2018). Earlier investigations have already revealed that ADSCs could be utilized for different human diseases. For example, human adipose-derived MSC (hADSC) effectively induced Treg cell generation and activation and regulated immune tol- erance in mice with collagen-induced arthritis, proposing that hADSCs are a good candidate for rheumatoid arthritis (RA) treatment (González et al. 2014). More importantly, ADSCs, induced by insulin-like growth factor 1 (IGF-1) and bone morphogenic protein 2 (BMP-2), could differentiate into chondrocyte-like cells that synthesized type II collagen and led to cartilage nodule formation (An et al. 2010). Fur- thermore, a recent study in rabbit model proved that ADSCs had ideal healing effects for full-thickness femoral articular cartilage defects (Mehrabani et al. 2015). Nonetheless, the efficacy of cartilage defect treatment using MSCs have usu- ally been limited by the fibroblast differentiation of chon- drocytes and the acquirement of treating with one or more growth factors (Deng et al. 2016). For maintenance of hya- line cartilage generated by stem cell-derived cartilage-like cells (chondroid cells), it is required to totally explain the molecular mechanisms regulating chondrogenic differentia- tion of ADSCs.

Accumulating evidences indicated that cytoskeleton and focal adhesion exerted significant effects on MSC differen- tiation (Mathieu and Loboa 2012; Steward and Kelly 2016). Cell adhesion provided a “permission” signal, allowing cell differentiation (Yeh et al. 2012). The chondrogenic differen- tiation of MSCs were mediated by various components and regulators of the ECM, such as collagen type II alpha 1 chain (COL2A1), aggrecan (ACAN), and SRY-box transcription factor 9 (SOX9), which were widely utilized as hyaline car- tilage marker genes (Annamalai et al. 2016; Fang et al. 2014; Liao et al. 2014). Integrin Subunit Beta 1 (ITGB1) is a non- tyrosine kinase collagen receptor protein and is needed for cell adhesion on ECM by interacting with collagen (Yeh et al. 2012). Because of its key roles in cell adhesion, ITGB1 has become an essential regulator of stem cell differentia- tion. For example, ITGB1 is highly expressed in neural stem
cells (NSCs), which supported NSC niche structural integ- rity to maintain hippocampal NSC population and regulated the astrocytic differentiation of hippocampal NSCs (Brooker et al. 2016). Furthermore, ITGB1 also promotes the osteo- blast differentiation and the formation of osteoblastic ECM in response to mechanical tensile strain (Zeng et al. 2015). More importantly, ITGB1 protein was lowly expressed in the bone marrow and adipose tissue-derived stem cells, but its expression was substantially increased during chondrogenic differentiation of these MSCs (Goessler et al. 2008). The ITGB1 signaling also regulated the hypertrophy of MSC chondrogenesis driven by deferral dynamic compression, via interaction with transforming growth factor beta 1 (TGF- β1) signaling (Zhang et al. 2015). Additionally, we reported earlier that ITGB1 expression was greatly increased in chondrogenic differentiation of ADSCs and decreased upon maturity (Luo et al. 2013). Nonetheless, the regulatory roles and molecular mechanisms of ITGB1 on chondrogenic dif- ferentiation of ADSCs are still largely unknown.

In this study, our objective was to study the molecular mechanisms underlying ITGB1-regulated ADSC chondro- genic differentiation by overexpressing ITGB1 in human ADSCs, followed by large-scale identification of differen- tially expressed genes via next-generation RNA sequenc- ing and label-free quantitative proteomics, providing new insights into the regulatory mechanisms of ADSC chon- drogenic differentiation, which might guide ADSC-based cartilage defect treatment.

Material and methods

Adipose tissue collection hADSCs were acquired from SALIAL Biotech Co., Ltd. (#saliai-HMSC (AD)-N, Guangzhou, China) and cultured in low-glucose DMEM (Gibco, USA) containing 10% FBS (#10099141C; Gibco, USA) and penicillin–streptomycin (100 mg/L) (Gibco, USA) at 37 °C in a humidified cham- ber with a supply of 5% CO2. The adherent cells in 6-well plates were subjected to cell identity confirmation by flow cytometry.

Flow cytometry

The identity of ADSCs isolated from human adipose tis- sues were confirmed by detecting the expression of cell surface markers CD45, CD73, CD90, and CD105 by flow cytometry (#cytoflex; Beckman Coulter, United States). Briefly, ADSCs acquired following three passages were then made into a single-cell suspension by digestion with trypsin (Sigma-Aldrich, United States). About 1 × 106 ADSC cells were mixed with 1 μL primary antibodies, specifically targeting CD45 (#MA5-17690, eBioscience, USA); CD73 (#12-0739-42, eBioscience, USA); CD90 (#17-0909-41,
eBioscience, USA); and CD105 (#MA1-19594, eBiosci- ence, USA) and incubated at 4 °C for 30 min, which were then analyzed using the flow cytometer. FlowJo 7.6 software analyzed the streaming results.

Cell transfection and treatments

To overexpress ITGB1 gene in ADSC cells, the coding sequences of ITGB1 gene were acquired by gene synthe- sis, which were then ligated using the LV003 vector. The LV003-ITGB1 recombinant plasmid was then presented into cultured ADSC cells by transfection using the Lipo- fectamine™ 2000 Reagent (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. The extracellular aggrecan was detected by Alcian blue staining (#G2542, Solarbio, China) following the manufacturer’s instructions. The gene overexpression efficacy was ultimately confirmed by measuring ITGB1 gene expression through quantitative qRT-PCR and Western blotting 48 h following cell transfec- tion. To inhibit ERK signaling, cells were treated with 5 nm SCH772984 (#HY-50846; MedChemExpress, China) for 24 h. Treatment with 10 nm GSK690693 (#S1113; Selleck, USA) for 24 h was applied to inhibit ATK signaling.

Quantitative RT‑PCR (qRT‑PCR)

The relative mRNA levels of the ITGB1 gene in ADSC cells were assessed using the quantitative RT-PCR (qRT-PCR) following cell transfection. Briefly, the total RNA samples of ADSC cells were isolated using the Trizol reagent (#R0016; Beyotime, Beijing, China) following the manufacturer’s instructions. About 3.0 μg RNA sample per cell group were subjected to synthesis of the cDNA library, using the Bestar qPCR RT kit (#2220; DBI Bioscience, Germany) following the manufacturer’s instructions. ITGB1 gene expressional levels were finally determined using real-time quantitative PCR method with the Bestar qPCR Master Mix kit (#2043; DBI Bioscience, Germany) following the manufacturer’s instructions. The quantitative PCR was conducted by pre- denaturation for 2 min at 94 °C, followed by 41 cycles of 94 °C for 20 s, 58 °C for 20 s, and 72 °C for 20 s. Relative mRNA levels were then measeured using the 2−ΔΔCt method as described earlier (Luo et al. 2013). GAPDH served as the internal standard. The sequences of primers utilized for quantitative RT-PCR were outlined in Table 1.

Western blotting

Total protein samples were extracted from cultured ADSC cells using the Tissue or Cell Total Protein Extraction Kit (#C510003; Sangon Biotech, China) following the instruction of the manufacturer. The protein concentra- tion was determined using the Modified Bradford reagent (#C100530; Sangon Biotech, China). Around 30 μg of pro- teins per cell group were then boiled at 100 °C for 5 min; separated with 10–12% SDS-PAGE; transferred onto 0.45 um PVDF membrane (Millipore, USA), which were then blocked with 5% lipid-free milk solution; incubated with primary antibodies (2 h at room temperature or overnight at 4 °C); incubated with horseradish peroxide reductase-con- jugated secondary antibodies; and finally developed with the enhanced chemiluminescence (ECL) substrates (Thermo Fisher Scientific, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal standard. The primary antibodies utilized in this study include anti- ITGB1 (#ab24693; Abcam, UK); anti-COL1A2 (#ab34712;Abcam, UK); anti-collagen type I alpha 1 chain (COL1A1) (#ab6308; Abcam, UK); anti-ACAN (#ab3778; Abcam, UK); anti-SOX9 (#82630; CST, USA); anti-p-AKT (#4060; CST, USA), anti-AKT (#9272; CST, USA); anti-ERK
(#ab32537; Abcam, UK); anti-p-ERK (#ab50011; Abcam, UK); and anti-GAPDH (#ab181602; Abcam, UK).

Next‑generation mRNA sequencing

Differentially expressed mRNA profiles in ADSCs induced by ITGB1 overexpression were characterized by large-scale next-generation deep sequencing meth- ods as described earlier with minor alterations (Oduor et al. 2017). Briefly, total RNAs were extracted from cul- tured ADSCs using the iPrep™ Trizol™ Plus RNA Kit (#IS10007; Thermo Fishier Scientific, USA) following the manufacturer’s instructions. Following RNA quality validation by agarose gel electrophoresis and OD260/280 value, the cDNA library was established with 0.2 μg RNA using the SmartPCR cDNA kit (#634925; CLONTECH Laboratories, Japan) following the manufacturer’s instruc- tions. After the removal of adaptor using RsaI digestion (Thermo Fisher Scientific, USA), cDNA samples were subjected to sonification fragmentation, profiling with Agilent Bioanalyzer, establishment of the Illumina library, and quality verification using an Agilent Bioanalyzer 2100 machine, which were finally sequenced by the Illumina HiSeq2000 system. Relative mRNA expressional levels were then determined using the Cufflinks 2.0.2 software. Significantly different expressed genes were defined by a fold change of > 2.0, a P value of < 0.01, and a false dis- covery rate (FDR) of < 0.05. Label‑free quantitative proteomics Differentially expressed proteins in ADSC cells were char- acterized by label-free quantitative proteomics with the use of liquid chromatography-mass spectrometry/mass spectrometry (LC–MS/MS) method as presented earlier with minor alterations (Li et al. 2017). Briefly, the total proteins of ADSC cells were extracted using the UA buffer containing 8 M urea and 150 mM Tris–HCl (pH 8.0) by centrifugation at 14,000×g for 20 min to remove cell debris. Next, protein solutions were incubated with 15 mM iodoacetamide for 30 min at room temperature in darkness and incubated with 25 mM NH4HCO3 for 30 min, fol- lowed by digestion with trypsin (Promega,USA) for 18 h at 37 °C. About 2 mg peptides were separated via the EASY- nLC1000 HPLC system (SC200 traps 150 μm × 100 mm RP-C18 Thermo EASY column; flow rate: 350 mL/ min). The MS/MS analysis was then conducted using a Q-Exactive mass spectrometer (Thermo Fisher Scientific, USA) with three biological replicates. Raw data from MS/MS analysis were processed using the MaxQuant software (version 1.5.2.8). Protein identification was finished by searching against the SwissProt Homo sapi- ens protein database. During the proteome database search, carbamidomethylation at cysteine residues was chosen as the fixed modification, while oxidation at methionine resi- dues and acetylation at protein N-terminal were chosen as the variable modifications. The FDR for peptide and protein identification was set as 0.01. Relative protein levels were determined by calculating the normalized spectral protein intensity levels. Differentially expressed proteins between ADSC cell groups were characterized by a FDR of < 0.01, a fold change of > 1.5 or < 0.667, and a P value of < 0.05. Bioinformatic analysis The hierarchical clustering plot, volcano plot, and scatter plot of differentially expressed genes or proteins were estab- lished with the use of the R software (1.0.8 version), based on relative mRNA or protein levels from abovementioned RNA sequencing and label-free quantitative proteomics. The enrichment of differentially expressed genes and proteins in signaling pathways was assessed using the Kyoto Ency- clopedia of Genes and Genomes (KEGG) platform (https:// www.genome.jp/kegg/). Statistical analysis The quantitative data in this study based on at least three biological repeats were presented as mean ± standard devia- tion (SD). The significant differences were assessed using Student’s T-test (two groups) and analysis of variance (ANOVA) methods as suitable. A P value of < 0.05 was considered statistically significant. Results ITGB1 overexpression promoted chondrogenic differentiation in hADSCs To study the molecular mechanisms regulating ADSC chon- drogenic differentiation, hADSCs had a significant expres- sion of the ADSC-specific surface marker proteins CD45, CD73, CD90, and CD105 in flow cytometry (Fig. 1a), con- firming the MSC identity of our isolated cells. To investigate the functions of ITGB1 in regulating ADSC chondrogenic differentiation, we overexpressed the ITGB1 gene in hAD- SCs by transfecting with recombinant LV003-ITGB1 plas- mid. The extracellular aggrecan was detected by Alcian blue staining, indicating that the blue points were significantly increased by ITGB1 overexpression in hADSCs, in com- parison with the negative control (Fig. 1b). Both ITGB1 mRNA and protein levels were also substantially increased following cell transfection (Fig. 2a, b). More importantly, we also noted that the expression of three hyaline cartilage marker genes COL2A1, ACAN, and SOX9 were substan- tially increased by ITGB1 overexpression in hADSCs, in comparison with the negative control (Fig. 2c). On the other hand, the expression of COL1A1 a fibrocartilage marker was substantially suppressed by ITGB1 overexpression in hADSCs, in comparison with the negative control (Fig. 2c), showing that we successfully isolated the human ADSC cells, in which ITGB1 overexpression effectively promoted its differentiation into chondrocytes. ITGB1 overexpression substantially modified mRNA expression profiles in hADSCs For insights into the molecular events underlying the regula- tion of hADSC chondrogenic differentiation by ITGB1 over- expression, we conducted a next-generation deep sequencing of mRNA expression in both hADSCs (negative control; the NC group) and ITGB1-overexpressing hADSCs (the ITGB1 group). Totally, we identified 246 genes, showing substantial differential expression in hADSCs induced by ITGB1 over- expression, such as 183 upregulated and 63 downregulated genes (Fig. 3a, b; Online Resource 1). Furthemore, differ- entially expressed genes induced by ITGB1 overexpression gene expression on hADSC surface was evaluated using flow cytom- etry. b The detection of extracellular aggrecan by Alcian blue staining in hADSCs were significantly enriched in a number of cel- lular signaling pathways, such as the PI3K-AKT signaling pathways, Ras and Rap1 signaling pathways, TNF signal- ing pathways, Toll-like receptor signaling, and many other pathways (Fig. 3c). Together, these findings proposed that the ITGB1 overexpression might promote the chondrogenic differentiation of hADSCs via regulation of distinct biologi- cal processes and signaling pathways. Fig. 1 The superficial phenomenon of hADSC by ITGB1 overexpres- sion. a Confirmation of hADSC identity by detecting the expression of surface marker genes CD45, CD73, CD90, and CD105. Marker ITGB1 overexpression induced remarkable proteomic profile changes in hADSCs For deep understanding of molecular mechanisms of ITGB1- regulated hADSC chondrogenic differentiation, we also characterized the significantly expressed proteins between hADSCs and ITGB1-overexpressing hADSCs in a large scale, through label-free quantitative proteomics based on LC–MS/MS method. Altogether, we identified 34 proteins, which were significantly differentially expressed in ITGB1- overexpressing hADSCs, in comparison with the normal hADSCs (Fig. 4a, b; Online Resource 2). Further informa- tion of these differentially expressed proteins were provided in Online Resource 2, including a number of cytoskeleton and cell adhesion-regulating proteins such as radixin (RDX), Taxilin Alpha (TXLNA), EH domain-containing protein 2 (EHD2), Catenin delta-1 (CTNND1), and mitogen-activated protein kinase 1/3 (MAPK1/3). In addition, these differen- tially expressed proteins were correlated with multiple sign- aling pathways, such as the mTOR signaling pathway, the estrogen signaling pathway, and signaling pathways involved in adherent junctions (Fig. 4c), showing that ITGB1 overex- pression caused significant changes in the proteomic profiles of hADSCs, which were correlated with different biological processes and cellular signaling pathways. Fig. 2 The molecular expres- sion of hADSC by ITGB1 overexpression. a, b Increased expression of ITGB1 gene in hADSCs transfected with recombinant LV-003-ITGB1 vectors. ITGB1 mRNA (a) and protein (b) levels were detected using quantitative RT-PCR and western blotting, respectively. c The expression of hyaline cartilage marker genes COL2A1, ACAN, and SOX9 and fibrocartilage marker gene COL1A1 in ITGB1- overexpressing hADSCs. Protein levels were detected using western blotting (left) and quantitated by normal- izing to GAPDH levels (right). hADSC human adipose-derived mesenchymal stem cell, ITGB1 transforming growth factor beta 1, NC negative control, GAPDH glyceraldehyde-3-phosphate dehydrogenase, COL2A1 colla- gen type II alpha 1 chain, ACAN aggrecan, COL1A1 collagen type I alpha 1 chain, SOX9 SRY-box transcription factor 9;**P < 0.01; **** < 0.0001. ITGB1 promoted hADSC chondrogenic differentiation by suppressing ERK signaling pathway As shown in prior transcriptome and proteome analysis, we discovered that the expression of key components of the AKT and ERK signaling pathways was greatly modified by ITGB1 overexpression (Figs. 3, 4; Online Resources 1 and 2), proposing that these two signaling pathways might be involved in the ITGB1-regulated hADSC chondrogenic differentiation. But with the merged analysis of all mRNAs and differentially expressed proteins from two omics, only one gene MAPK3 involved in ERK signaling are present in both (Online Resource 3). To test this, we first detected the abundances of AKT and ERK proteins, as well as their phosphorylation, between hADSCs and ITGB1-overexpressing hADSCs and discovered that the phosphorylation of AKT and ERK pro- teins were substantially suppressed by ITGB1 overexpres- sion in hADSCs (Fig. 5a). Treatment with ERK inhibitor SCH772984 greatly suppressed ERK phosphorylation in hADSCs, and the COL2A1, ACAN, and SOX9 protein levels in SCH772984-treated hADSCs were substantially higher than those of the hADSCs with no SCH772984 treatment (Fig. 5b). Furthermore, SCH772984 treatment caused even more significant increase of COL2A1, ACAN, and SOX9 proteins in SCH772984-treated ITGB1-over- expressing hADSCs, in comparison with ITGB1-overex- pressing hADSCs with no SCH772984 treatment (Fig. 5b). The expression of COL1A1 was not substantially modi- fied by SCH772984 treatment in ITGB1-overexpressing hADSCs (Fig. 5b). However, treatment with AKT inhibi- tor GSK690693 failed to increase COL2A1, ACAN, and SOX9 expression in hADSCs but substantially increased COL1A1 expression in hADSCs (Online Resource 4). Altogether, these findings proved that ITGB1 promoted the chondrogenic differentiation of hADSCs via the suppres- sion of the ERK signaling pathway, other than the AKT signaling pathway.

Fig. 3 Differential mRNA profiles induced by ITGB1 overexpres- sion in hADSCs. a The hierarchical clustering of significantly dif- ferent expressed mRNAs in hADSCs with ITGB1 overexpression. High and low gene expressions were indicated by red and green lines, respectively. b A scatter-plot representation of significantly differen- tial expressed mRNAs between hADSCs and ITGB1-overexpressing

Discussion

Stem cells are a promising strategy for treatment of cartilage defects (Deng et al. 2016; Nam et al. 2018). Among the many types of stem cells, the application of embryonic stem cells has been greatly limited by the widely recognized ethi- cal concerns (Stromps et al. 2014). Hence, MSCs are benefi- cial options for stem cell-based cartilage defect treatments, hADSCs. c The KEGG signaling pathways with substantial enrich- ments of differentially expressed genes hADSCs due to ITGB1 overexpression. hADSC human adipose-derived mesenchymal stem cell, ITGB1 transforming growth factor beta 1, NC negative control,KEGG Kyoto Encyclopedia of Genes and Genomes especially the BMSCs and hADSCs because of their high chondrogenic potential (Nam et al. 2018). More importantly, the harvesting of ADSCs is less invasive but with higher yield in comparison with BMSCs, which make ADSCs more preferable for cartilage defect treatment (Nam et al. 2018). Nonetheless, the regulatory mechanism of ADSC chon- drogenic differentiation is still poorly understood. In the current study, we successfully isolated MSCs from human with a significantly different expression in hADSCs induced by ITGB1 overexpression. c The KEGG pathway analysis of differen- tially expressed proteins between hADSCs and ITGB1-overexpress- ing hADSCs. hADSC human adipose-derived mesenchymal stem cell, ITGB1 transforming growth factor beta 1, NC negative control, KEGG Kyoto Encyclopedia of Genes and Genomes adipose tissues and overexpressed the ITGB1 gene in these ADSCs, which resulted into a greatly increased expres- sion of hyaline cartilage marker genes such as COL2A1, ACAN, and SOX9. By combining the transcriptome analy- sis and proteome analysis, we characterized a large number of differentially expressed genes and proteins induced by ITGB1 overexpression in ADSCs, which were correlated with different biological processes and signaling pathways. Furthermore, we ultimately proved that ITGB1 promoted ADSC chondrogenic differentiation by suppressing the ERK signaling pathway. These findings provided novel insights into the molecular mechanisms of ADSC chondrogenic differentiation, which might hopefully guide the ADSC- based cartilage defect treatment.

Fig. 4 Differentially expressed proteins caused by ITGB1 overex- pression in hADSCs. a The hierarchical clustering of differentially expressed proteins between hADSCs and ITGB1-overexpressing hADSCs. Differential expression of proteins identified by label-free quantitative proteomics were indicated by red (high expression) or blue (low expression) bars. b A volcano plot representing proteins

Fig. 5 ITGB1 suppressed ERK signaling to promote hADSC chondrogenic differentiation.a The abundances of AKT and ERK proteins and their phos- phorylation regulated by ITGB1 overexpression in hADSCs.Protein levels in hADSCs were detected using western blotting, with GAPDH as the internal standard. b Regulation of ERK phosphorylation and COL2A1, COL1A1, ACAN, and SOX9 protein expression by ERK inhibitor SCH772984. hADSCs and ITGB1-overexpressing hADSCs were treated with 5 nm SCH772984 for 24 h, followed by western blotting analysis. hADSC human adipose-derived mesenchymal stem cell, ITGB1 transforming growth factor beta 1, NC negative control, COL2A1 collagen type II alpha 1 chain, ACAN aggrecan, COL1A1 collagen type I alpha 1 chain, SOX9 SRY-box transcrip- tion factor 9, GAPDH glyceral- dehyde-3-phosphate dehy- drogenase, ERK extracellular regulated protein kinase, p-ERK phosphorylated ERK, AKT pro- tein kinase B, p-AKT phospho- rylated AKT, SCH SCH772984; * < 0.05; ** < 0.01; *** < 0.001;**** < 0.0001. Prior induction of stem cell differentiation toward desired cell types has been chiefly dependent on the application of different growth factors (Chang et al. 2006). The discovery of cell adhesion and mechanical stimulation as potent regu- latory factors controlling MSCs has provided more options to effectively regulate stem cell differentiation in future clini- cal application (Wang and Chen 2013). It is well-known that ITGB1, among the major members of collagen recep- tor, exerted important effects on cell adhesion formation by promoting the interaction of cells with the ECM (Davy and Robbins 2014). Consistent with this, ITGB1 regulates the differentiation of different stem cell types, as shown in the Introduction section. Nonetheless, its roles in chondro- genic differentiation of MSCs from adipose tissues are still largely unexplored. In this study, we demonstrated that the overexpression of ITGB1 gene in ADSCs greatly modi- fied the expression levels of different hyaline cartilage and fibrocartilage markers genes. Among them, COL2A1 could enhance the chondrogenic differentiation of human bone marrow-derived MSCs (Annamalai et al. 2016). Aggrecan is a major chondrogenic ECM gene, and SOX9 has also a potential in chondrogenic differentiation (Fang et al. 2014; Liao et al. 2014). The expression of COL2A1, ACAN, and SOX9 has been widely utilized as markers for chondrogenic differentiation and hyaline cartilage, which were all remark- ably increased by ITGB1 overexpression in ADSCs. In con- trast, the expression of collagen I, one major fibrocartilage marker (Zhuo and Chen 2016), was greatly suppressed by ITGB1 overexpression in ADSCs, persuasively revealing the chondrogenic differentiation-promoting roles of ITGB1 in ADSCs. Through the combination of next-generation RNA sequencing and label-free quantitative proteomics, we char- acterized a large number of differentially expressed genes and proteins in ITGB1-overexpressing ADSCs. We noticed that numerous differentially expressed genes were involved in cytoskeleton and cell adhesion such as CTNND1 and RDX (Hamada et al. 2014; Yang et al. 2016), which is con- sistent with the biochemical functions of ITGB1 as a colla- gen-interacting protein. The earlier study indicated that the actin cytoskeleton-linking adaptor proteins RDX could regu- late cell–cell adhesion in prostate cancer cells (Ferran et al. 2012). On the other hand, in this study, RDX expression was substantially downregulated by ITGB1 overexpression in ADSCs (Online Resource 2), proposing that this protein might also regulate the ADSC chondrogenic differentiation through modulation of stem cell adhesion, which deserves further studies. Furthermore, numerous proteins that are closely correlated with cell differentiation were differen- tially expressed proteins induced by ITGB1 in this study. For example, the MAPK signaling pathway was among the key regulatory mechanisms of cell differentiation and other cellular processes (Sun et al. 2015; Upadhya et al. 2013). MAPK1 and MAPK3 expressions were both greatly modi- fied in ITGB1-overexpressing ADSCs (Online Resource 2). Further functional studies of these differentially expressed proteins would deepen our understanding of stem cell chon- drogenic differentiation. Finally, we examined the mediating roles of the ERK signaling pathway in ITGB1-driven ADSC chondrogenic differentiation based on our omics data. The ERK signaling pathway was earlier correlated with different aspects of stem cell biology (Baumgartner et al. 2018; Chan et al. 2018). For example, recent reports proved that the activation of ERK signaling mediated the osteogenic differentiation of human bone marrow-derived MSCs induced by Naringin (Wang et al. 2017). In ADSCs, the ERK signaling pathway mediated their differentiation into endothelial cells induced by growth factors (Almalki and Agrawal 2017). In the current study, we revealed that the ERK signaling pathway was greatly suppressed in ITGB1-induced ADSC chondrogenic differ- entiation. To confirm the mediating roles of ERK signaling in this process, we apply the SCH772984, a specific and potent inhibitor of the ERK1/2 proteins (Bian et al. 2016). SCH772984 together with ITGB1 overexpression induced a remarkably increased expression of abovementioned hyaline cartilage marker genes, which further supported the roles of ERK in ADSC chondrogenic differentiation. In a nutshell, we demonstrated in this study that ITGB1 overexpression substantially promoted the chondrogenic differentiation of MSCs from human adipose tissues. Our transcriptome and proteomics analysis revealed different biological processes and signaling pathways regulated by ITGB1 during ADSC chondrogenic differentiation. ITGB1- induced ADSC chondrogenic differentiation was mediated by its suppression on ERK signaling pathways, revealing new mechanisms that regulate the differentiation of ADSCs into chondrocytes, which provided a basis for ADSC-based treatment of cartilage defects. Author contributions ZZ conceived and designed the study, and criti- cally revised the manuscript. SL performed the experiments, analyzed the data and drafted the manuscript. QS, WL and WW participated in study design, study implementation and manuscript revision. All authors read and approved the final manuscript. Funding This study was funded by the National Natural Science Foun- dation of China (Grant Number 81472089); and Guangdong Provincial Medical Scientific Research Foundation (Grant Number A2018299, B2019038). Data availability The datasets generated during and/or analysed dur- ing the current study are available from the corresponding author on reasonable request. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. References Almalki SG, Agrawal DK (2017) ERK signaling is required for VEGF- A/VEGFR2-induced differentiation of porcine adipose-derived mesenchymal stem cells into endothelial cells. Stem Cell Res Ther 8:113 An C, Cheng Y, Yuan Q, Li J (2010) IGF-1 and BMP-2 induces dif- ferentiation of adipose-derived mesenchymal stem cells into chondrocytes-like cells. 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