BAG3 regulates bone marrow mesenchymal stem cell proliferation by targeting INTS7

Cell culture and transfection

The OriCell Strain C57BL/6 mouse BMMSCs were purchased from Cyagen Biosciences (Guangzhou, China) and were cultured in OriCell C57BL/6 mouse BMMSC complete medium containing 10% fetal bovine serum (FBS), 1% glutamine, and 1% penicillin-streptomycin at 37 °C in a humid 5% CO₂ atmosphere. The phenotype, pluripotency, and stem cell properties of the BMMSCs were evaluated as previously reported (Chen et al., 2020; Liu et al., 2013; Liu et al., 2021). The BAG-targeting small interfering (si)RNA (si-BAG3) or the negative control siRNA (si-NC) were transfected into BMMSCs using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA), according to the manufacturer’s instructions. The nucleotide sequences of the BAG-targeting siRNAs were as follows: si-BAG3 #1, 5′-AAGAUGCAGUGUCCUUAGG-3′; si-BAG3 #2, 5′-UUGGCUUCUAGCUGUUGGC-3′. The nucleotide sequence of the si-NC was described previously (Xue et al., 2022). Additionally, the pcDNA3.1-BAG3, pcDNA3.1-INTS7, and empty vector (pcDNA3.1-NC) were purchased from GenePharma (Suzhou, China) and used to transfect BMMSCs in Opti-minimum essential medium (MEM; Invitrogen, Carlsbad, CA, USA) using the Lipofectamine 2000. Cells were harvested for analysis 48 h post-transfection.

RNA extraction and quantitative real-time(qRT)-PCR

To further assess the expression of target genes in the transfected cells, total RNA was extracted from transfected cells using the RNeasy Plus Micro Kit (Qiagen, Düsseldorf, Germany), according to the manufacturer’s instructions. A PrimeScript Reverse Transcription Kit (Vazyme, Nanjing, China) was then used for reverse transcription, and the SYBR Premix Ex Taq polymerase (Takara, Dalian, China) were used to conduct the qRT-PCR on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). 18S ribosomal (r)RNA was used as an endogenous control and expression was calculated using the 2^−ΔΔCt method. The following primers were used in the qRT-PCR reaction: BAG3 forward 5′-CCGGGCTGGGAGATCAAAAT-3′ and reverse 5′-GGCTGAAGATGCAGTGTCCTTA-3′; and 18S rRNA forward 5′-AAACGGCTACCACATCCAAG-3′ and reverse 5′-CCTCCAATGGATCCTCGT TA-3′.

Cell proliferation assays

Cell proliferation was detected using the Cell Counting Kit-8 (CCK-8) (Beyotime, Nantong, China) at four time points (0, 24, 48, 72, and 96 h), in accordance with the manufacturer’s protocol. Briefly, after transfection for 48 h, 4,000 cells/well were seeded into 96-well plates. The CCK-8 solution was then added to each well and incubated at 37 °C for another 2 h. The optical absorbance of each well was read at 450 nm using a microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA).

For the colony-formation assay, cells were plated in 6-well plates at a density of 1,000 cells/well and cultured for 2 weeks, before harvesting. Colonies were fixed with methanol and stained with 0.1% crystal violet dye (Beyotime). The number of cell colonies was observed and counted using a bright-field microscope.

Transwell migration assay

Migration was assessed using the transwell migration assay, as reported previously (Wang et al., 2022; Xu et al., 2022; Xue et al., 2022; Zhou et al., 2022b). Briefly, after transfection for 48 h, 3 × 10⁴ cells in 300 µL of serum-free medium were seeded into the upper chamber of each well of a 24-well plate (Merck Millipore), while 700 µL complete medium was added to the lower chamber. After culture for 24 h, cells in the lower chamber were collected, then fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for counting.

Cell cycle and apoptosis assays

For cell cycle analysis, BMMSCs were fixed in 70% ethanol at 4 °C for 48 h and then stained with propidium iodide (PI). Thereafter, the relative content of intracellular DNA was measured by flow cytometry (BD Biosciences, Heidelberg, Germany).

Cellular apoptosis was assayed using an Apoptosis Detection Kit (Vazyme), as previously mentioned (Xue et al., 2022; Zhao et al., 2019; Zhou et al., 2022a). Briefly, cells were fixed in methanol and treated with 0.1% Triton X-100 for 10 min at room temperature, followed by BrightRed Labeling Buffer for 60 min at 37 °C. After washing with phosphate buffered saline (PBS) three times, the samples were stained with DAPI. Images were captured using a confocal laser microscope (Zeiss LSM800; Carl Zeiss, Oberkochen, Germany).

Co-immunoprecipitation (Co-IP) assay

Co-IP assay was performed by incubating cell lysates with an anti-BAG3 antibody (Proteintech, Chicago, IL, USA) or an anti-INTS7 antibody (Proteintech), as previously described (Xue et al., 2022). Briefly, the cells were lysed on ice for 30 min using RIPA lysis buffer (Beyotime, Nantong, China) and then precleared with protein A agarose(Invitrogen). The lysates were incubated with the indicated antibodies overnight at 4 °C and then with protein A agarose at 4 °C for 3 h. The immunoprecipitants were washed with PBS, eluted with sodium dodecyl sulfate buffer, and analyzed by western blotting.

Protein-protein interaction

BAG3 and INTS7 sequences (FASTA format) were obtained from the National Center for Biotechnology Information (NCBI). Homology modeling analysis was done through the Robetta Modeling Server (https://robetta.bakerlab.org). The output is a three-dimensional (3D) protein model in Protein Data Bank (PDB) format. The Robetta server identified hot residues with 79% accuracy by calculating parameters such as implicit solventization and hydrogen bonding, stacking interactions, solventization interactions, and Lennard-Jones interactions. We then predicted the BAG3-INTS7 interaction interface using HawkDock and visualized it using PyMOL (https://pymol.org/2/).

Western blotting

Western blotting was performed as previously described (Xue et al., 2022; Yu et al., 2022). In brief, cell lysates were isolated on an SDS-PAGE gel and then transferred onto polyvinylidene difluoride membranes(Millipore, Bedford, MA, USA). The membranes were blocked in Tris-buffered saline tween (TBST) containing 5% fat-free milk for 1 h at room temperature, and incubated with the indicated dilutions of primary antibodies (anti-BAG3, 1:500; anti-INTS7, 1:1,000; and anti-tubulin, 1:1,000; all from Proteintech) at 4 °C overnight. The next day, the membranes were washed and treated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. Western blots were developed and visualized using the Enhanced Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA). Tubulin was used as a loading control. For protein half-life assay, BMMSCs was incubated with 100 µg/mL cycloheximide (CHX) to block protein synthesis, and proteins were assayed after 0, 4 and 8 h.

ROS analysis

After transfection, cells were incubated with 20 µM 2′, 7′-dichlorofluorescein diacetate (DCFDA; Invitrogen, Waltham, MA, USA) for 40 min at 37 °C. DCFDA is a fluorogenic dye, which is used for the measurement of hydroxyl, peroxyl, and other ROS activity within cells. Fluorescence intensity was observed using a confocal laser microscope (Zeiss LSM800; Carl Zeiss), according to our previously reported methods (Liu et al., 2021).

Statistical analysis

GraphPad Prism 9.0 (GraphPad, San Diego, CA, USA) and SPSS 22.0 software (IBM, Chicago, IL, USA) were used for data analysis. All experiments were repeated at least three times. All quantitative data are expressed as the mean ± standard deviation (SD). The Student’s t-test and the one-way ANOVA were used to calculate p-values. All p-values were two-sided, and the threshold for statistical significance was set at p < 0.05.

BAG3 promotes BMMSCs expansion in vitro

To investigate the effects of BAG3 on BMMSCs growth, we transfected BMMSCs with two BAG3-targeting siRNAs (si-BAG3 1# and si-BAG3 2#) to inhibit BAG3 expression. The qRT-PCR and western blots results showed that the expression of BAG3 was significantly lower in BMMSCs transfected with the two BAG3-targeting siRNAs, than in BMMSCs transfected with the negative control siRNA (si-NC) (Figs. 1A–1C). As expected, we observed a significant decrease in the proliferation and colony formation capacity of the BMMSCs transfected with si-BAG3, but not those transfected with si-NC, in the CCK-8 and colony formation assays (Figs. 1D–1F). Moreover, the transwell migration assay results showed a significant decrease in the number of migrating BMMSCs after BAG3 downregulation (Figs. 1G and 1H). To further assess whether BAG3 silencing in BMMSCs had an effect on the cell cycle and the rate of their apoptosis, a flow cytometry and TUNEL analysis was performed. Flow cytometry exhibited that BAG3 silencing delayed the progression of cell cycle and inhibited cell proliferation by arresting BMMSCs at G0/G1 phase (Figs. 1I and 1J). We also found that the reduction in BAG3 expression increased the number of apoptotic BMMSCs (Figs. 1K and 1L).

To further validate the role of BAG3 in BMMSCs, cells were transfected with pcDNA3.1-BAG3 or vector control. Western blot assays showed that the expression of BAG3 was increased in BAG3 overexpression cells (Figs. 2A and 1B). Moreover, cell growth was markedly increased after BAG3 overexpression in BMMSCs (Figs. 2C–2E). Meanwhile, an increase in cell migration was observed following the transfection of BMMSCs with pcDNA3.1-BAG3, compared with the migration of BMMSCs transfected with empty vector (Figs. 2F and 1G). These data collectively indicate that BAG3 promoted the expansion and migration of BMMSCs, while inhibiting their apoptosis.

BAG3 interacts with INTS7

In our previous study, the endogenous INTS7 complexes purified from the BMMSCs by IP were analyzed by MS (Liu et al., 2021) The results showed that BAG3 was a potential interaction partner of INTS7. In the present study, we first predicted the three-dimensional structure of the BAG3-INTS7 complex using the Robetta web server (https://robetta.bakerlab.org/) (Yang et al., 2020) (Fig. 3A). We then predicted the BAG3-INTS7 interaction interface using HawkDock and visualized it using PyMOL (https://pymol.org/2/) (Weng et al., 2019) (Fig. 3B). The key residues predicted to be involved in the BAG3-INTS7 interaction are listed in Table S1. Next, a BAG3-INTS7 Co-IP experiment was performed to support these bioinformatics models. The results confirmed the protein interaction between BAG3 and INTS7 in BMMSCs (Fig. 3C). CHX treatment reveals a significant reduction in INTS7 protein half-life after BAG3 interference (Figs. 3D and 3E). Additionally, the western blotting results showed that BAG3 silencing reduced INTS7 protein expression, while treatment of MG132(proteaosomal inhibitor) could rescue destabilized INTS7 expression following BAG3 knockdown (Figs. 3F and 3G). These results indicate that BAG3 was indeed directly binding to INTS7 and affecting its stability.

Ubiquitination marks proteins for degradation (Van Wijk et al., 2019). To investigate whether ubiquitination was involved in the BAG3-mediated regulation of INTS7 stability, we conducted ubiquitination experiments. As shown in Fig. 3H, a significant increase in INTS7 ubiquitination was detected following the transfection of BMMSCs with si-BAG3 but not si-NC. These results show that BAG3 stabilized INTS7 and prevented it from being marked for degradation by ubiquitination.

BAG3 silencing triggers oxidative stress

ROS regulate the survival, proliferation, and terminal differentiation of mesenchymal stem cells (MSCs) (Liu et al., 2021). In general, low levels of ROS promote MSC proliferation, differentiation, and survival, while elevated ROS levels (defined as oxidative stress) induce MSC apoptosis. Moreover, our previous study reported that INTS7 and its binding protein ABCD3 affect ROS levels (Liu et al., 2021). We anticipated that BAG3, as an important binding partner of INTS7, could affect BMMSC proliferation via a similar mechanism. To investigate whether oxidative stress inhibition was involved in the BAG3-INTS7-mediated regulation of BMMSCs expansion, we examined ROS and antioxidant levels in BMMSCs after BAG3 silencing. In addition, DNA damage is associated with oxidative stress induction. The detection of nuclear γ-H2AX foci, a biomarker for DNA double-strand breaks, provides indirect evidence of oxidative stress (Sangermano et al., 2019). We found that both the ROS levels (Figs. 4A and 4B) and the percentage of γ-H2AX-positive cells (Figs. 4C and 4D) were all significantly increased in BMMSCs transfected with si-BAG3 vs. si-NC. These findings suggest that oxidative stress was activated in BAG3-deficent BMMSCs.

ROS regulate the BAG3-INST7-induced proliferation and migration of BMMSCs

To further investigate whether ROS altered the phenotype of BMMSCs with reduced BAG3 expression, we conducted rescue experiments by co-transfecting BMMSCs with pcDNA3.1-INTS7 and the antioxidant scavenger, N-acetylcysteine (NAC), respectively. As expected, pcDNA3.1-INTS7 and NAC reversed the effect of BAG3 silencing on the proliferation (Fig. 5A), and colony formation (Figs. 5B and 5C) and migration capacities of BMMSCs (Figs. 5D and 5E). These results imply that ROS levels regulate the INST7-BAG3-mediated expansion of BMMSCs.