PDGF‐C promotes cell proliferation partially via downregulating BOP1

Platelet‐derived growth factor C (PDGF‐C) is a member of PDGF/VEGF family, which is well‐known for important functions in the vascular system. It is widely reported that PDGF‐C is able to modulate cell proliferation. However, it is still not very clear about this cell modulating mechanism at the molecular level. In a screening of factors regulated by PDGF‐C protein, we fished out a factor called block of proliferation 1 (BOP1), which is a pivotal regulator of ribosome biogenesis and cell proliferation. In this study, we investigated the regulation of BOP1 by PDGF‐C and its role in modulating cell proliferation. We found that BOP1 was downregulated at both mRNA and protein levels in cells treated with PDGF‐C‐containing conditioned medium. On the other hand, BOP1 was upregulated in PDGF‐C deficient mice. Furthermore, we confirmed that overexpression of BOP1 inhibited HEK293A cell proliferation, whereas knockdown of BOP1 promoted cell proliferation. The mitogenic effect of PDGF‐C could be attenuated by downregulation of BOP1. Our results demonstrate a clear PDGF‐C–BOP1 signaling that modulates cell proliferation.

At the cellular level, PDGF-C was identified as a potent mitogen when it was identified (Hamada et al., 2000).After it is secreted extracellularly in a paracrine fashion, PDGF-C could bind to PDGF receptor alpha (PDGFRα) (Fredriksson et al., 2005).Although currently much is known about the downstream of the PDGF-C pathway, some of which may be related to its mitogenic function.It is important to understand the downstream mechanisms underlying PDGF-C mitogenic functions.
To explore the molecular mechanism of PDGF-C mitogenic functions, we scrutinized our published microarray data of factors modulated by the PDGF-C protein (Tang et al., 2010).Among these candidates, we identified a factor called block of proliferation 1 (BOP1) which may mediate the functions of PDGF-C in modulating cell proliferation.BOP1 is a member of the PES1-BOP1-WDR12 (PeBoW) complex, which plays critical roles in the ribosomal RNA processing (Rohrmoser et al., 2007;Strezoska et al., 2000).It was initially reported to be an inhibitor of cell proliferation by interfering with multiple reactions in pre-RNA processing in the cells (Holzel et al., 2005;Strezoska et al., 2002).However, subsequently it was discovered that BOP1 could also promote cell proliferation in some cancer cells (Vellky et al., 2021).

C Cell ell B Biology iology I International nternational
This study was designed to test the hypothesis that the mitogenic effects of PDGF-C is mediated through a pathway involving the regulation of BOP1.

| Cell culture
HEK293A cells from the American Type Tissue Collection were cultured in Dulbecco's modified Eagle medium (ExCellBio) supplemented with 10% fetal bovine serum (FBS; ExCellBio) and 1% penicillin and streptomycin (Cellgro) at 37°C in a 5% CO 2 + 95% air incubator.Cells were passaged 1 day before they were subjected to various treatments.

| Transfection
Plasmids and siRNA were transfected into HEK293A cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols.In brief, plasmids or siRNA were mixed with Lipofectamine 2000 in Opti-MEM medium and incubated for 15 min at room temperature for the nucleic acid-Lipofectamine complex formation.
The complex was then added to cell cultures in a complete culture medium as described above.Cells were then incubated for 48 h.In this study, the transfection efficiency was approximately 90%.

| Collection of conditioned medium
Two days after HEK293A cells were transfected, the cells were washed in phosphate-buffered saline (PBS) and then maintained in serum-free medium which was then collected 6 h later to be used as conditioned medium (CM) after removing of debris by centrifugation.

| Western blot
Cultured HEK293A cells were lysed in radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific).After that, they were quantitated with a DC™ Protein Assay Kit (Bio-Rad), separated in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis matrix, and transferred to a polyvinylidene difluoride membrane (Bio-Rad).
After a brief Ponceau S (p3504; Sigma) staining, the membrane was blocked with 5% bovine serum albumin, followed by incubation with a primary antibody at 4°C overnight, and incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) for 1 h at room temperature.Each incubation was followed by three washes with Tris-buffered saline buffer with Tween-20.Two sets of primary antibodies, mouse anti-BOP1 (sc-390672, 1:1000; Santa Cruz) and rabbit anti-β-tubulin (AP0064, 1:1000; Bioworld), were used in this study.Immobilon Western Chemiluminescent HRP substrate (Merck Millipore) was used to detect specific antibody binding.Signals were captured using a G-BOX (Syngene) imaging system.To determine the relative expression of a certain protein, ImageJ (NIH) software was used to quantify the relative expression level of the protein, followed by normalizing to the β-tubulin housekeeping control.

| Cell proliferation assay
HEK293A cells were cultured in 96-well plates.The cell viability was determined by the cell counting kit-8 (K1018; APExBio) staining at 48 h after transfection.It is a convenient and sensitive method for cell density measurement.The absorbance of each well was measured by a microplate reader at 450 nm and analyzed as described in the protocol from the company.
The expression of BOP1 was normalized to the level of β-actin in the same cDNA.The ∆∆ 2 Ct method was used to compare mRNA expression levels.

| Statistical analysis
The statistical analysis was performed using the GraphPad Prism 8.0 software.All data were tested for normality and equal variance.If the data passed these tests, the two-group comparison was evaluated by Student's t-test and one-way or two-way analysis of variance.If the data did not pass these tests, Mann-Whitney test was performed to compare the two groups.All quantitative data in this study was presented as mean ± standard error of mean, and p < .05 was considered statistically significant.Each experiment was repeated at least three times.

| PDGF-C promotes cell proliferation
PDGF-C was identified as a potent mitogen when it was identified (Hamada et al., 2000).To study the functions, we established a cell Then we applied the serum-free CM to another passage of HEK293A cells and measured their proliferation.As shown in Figure 1c, the cells cultured with PDGF-C protein containing CM demonstrated an approximately one-third higher rate of proliferation than the cells cultured with the control CM.This result is consistent with our hypothesis that secreted PDGF-C promotes cell proliferation in this cell culture system.

| PDGF-C downregulates BOP1 expression but does not alter its subcellular localization
Next, we explored the molecular mechanism of PDGF-C mitogenic effects.In a previously study, we performed a microarray study of retina treated with PDGF-C protein (Tang et al., 2010).Data was deposited in the Gene Expression Omnibus under accession No.
GSE19207.In this data, we focused on BOP1 as a candidate because it is a regulator of cell proliferation.In the microarray study, Bop1 was downregulated by 1.45 fold in the retina treated with PDGF-C protein (GSE19207).
To confirm the possibility that BOP1 is a downstream factor of PDGF-C signaling, we examined the expression of BOP1 in HEK293A cells after cultured with PDGF-C containing CM. BOP1 expression was examined at both mRNA and protein levels.Quantitative realtime PCR (qPCR) study indicated that the expression level of BOP1 mRNA was downregulated in cells cultured with PDGF-C containing CM (Figure 2a).Western blot study confirmed that BOP1 protein was significantly downregulated in cells treated with secreted PDGF-C protein (Figure 2b).We further examined other possible effects of PDGF-C signaling on BOP1.To follow the localization of BOP1 after the application of PDGF-C CM.We found BOP1 protein was normally located in the nucleoli which were not counterstained with DAPI (Figure 2c).In the cells cultured with PDGF-C CM, the localization of BOP1 was not altered.
Therefore, these results demonstrate that PDGF-C downregulates BOP1 expression but does not alter its localization.

| pdgf-c Deficiency upregulates BOP1 expression
To further confirm that BOP1 is a downstream effector of PDGF-C signaling, we examined the expression of BOP1 in pdgf-c deficient mice.Both PDGF-C and BOP1 are predicted (https://www.ncbi.nlm. nih.gov/gene) to be highly expressed in skeletal muscles.Thus, skeletal muscles in the mouse posterior limb were isolated for the study of BOP1 expression.qPCR study showed that the expression level of BOP1 was upregulated in pdgf-c deficient mice at the mRNA level (Figure 3a).Western blot verified that BOP1 protein was also significantly upregulated in pdgf-c deficient mice (Figure 3b).These results indicate that pdgf-c deficiency upregulates BOP1 expression.
It confirms that PDGF-C downregulates BOP1 expression.

| BOP1 inhibits HEK293A cell proliferation
Our results indicated that the expression of BOP1 is suppressed by PDGF-C.Therefore, the effects of BOP1 downregulation on cell proliferation was studied.BOP1 was shown to be a cell proliferation inhibitor for NIH 3T3 cells (Pestov et al., 1998).However, in some cancer cell lines, it could instead promote cell proliferation (He et al., 2022).
To examine the effects of BOP1 on cell proliferation in our culture system, we modulated the expression of BOP1 in HEK293A cells and examined their proliferation.Western blot confirmed that BOP1 was successfully regulated at the protein level (Figure 4a).Cell proliferation decreased significantly in the BOP1 overexpression group, while increased significantly in the BOP1 knockdown group (Figure 4b).These results confirm that BOP1 is an inhibitor of cell proliferation in HEK293A cells.effects of PDGF-C CM in promoting cell proliferation under physiological expression level of BOP1 was about 35%.And it dropped to 11% in BOP1 knockdown condition.This result suggests that BOP1 partially modulates the mitogenic effects of PDGF-C, but it does not provide the only pathway.

| DISCUSSION
In this study, we examined the downregulation of BOP1 by PDGF-C, and confirmed that the downregulation of BOP1 protein promoted cell proliferation.These results demonstrate PDGF-C-BOP1 signaling is able to modulate cell proliferation.In addition, the study of PDGF-C CM on BOP1 transfected cells suggests that BOP1 is one of the multiple downstream mitogenic effectors of PDGF-C.Taken together, we demonstrate that PDGF-C promotes cell proliferation partially via downregulating BOP1 expression.
We expressed PDGF-C protein in HEK293A cells, collected CM, and examined its effects on another passage of HEK293A cells.There may be differences between CM and the PDGF-C protein as the CM also contains other factors.In our CM system, these additional factors could be introduced by the overexpression of PDGF-C.PDGF-C is an important factor involved in many biological processes.It was discovered more than 20 years ago (Li et al., 2000).
The main significance of this study is that we identified a novel pathway that links PDGF-C signaling with chromosomal stability because the downstream factor BOP1 is the member of the PeBoW complex which has close relationship with chromosomal instability (Rohrmoser et al., 2007;Strezoska et al., 2000).BOP1 was initially identified as a proliferation inhibitor in an isolation of growth inhibitory sequences from a cDNA library (Pestov et al., 1998).And the assay of the initial study on BOP1 was performed on NIH 3T3 cells.Its proliferation inhibitory function could be confirmed both in our study on HEK293A cells and the study on human aortic smooth muscle cells (Wu et al., 2019).
Another significance of this study is that we identified PDGF-C as an extracellular modulator of BOP1.Because of the limitation in BOP1 study, as we know, there is no report about an extracellular factor to modulate BOP1.The functions of BOP1 in cancer cell proliferation is different from our results.In cancer cells, BOP1 promotes cell proliferation, epithelial-to-mesenchymal transition, tumorigenesis, and cancer development (Chung et al., 2011;He et al., 2022;Vellky et al., 2021).In this aspect, as a modulator of BOP1, PDGF-C and its receptor may be the targets for modulating cancer cells.The significance of PDGF-C in modulating cancer cells via regulating BOP1 should be further investigated.
The mechanism of BOP1 in inhibiting proliferation may be based on its role in the PeBoW complex.BOP1 is basically a ribosome biogenesis protein.At the molecular level, BOP1 forms the nucleolar PeBoW complex together with Pes1 and WDR12, which are also essential for cell proliferation (Grimm et al., 2006).Also, BOP1 is related to chromosomal instability (Lips et al., 2008).The basic function of the PeBoW complex require the maturation of 25S and 5.8S ribosomal RNAs and 60S ribosome biogenesis (Pestov et al., 2001;Strezoska et al., 2000).In Saccharomyces cerevisiae, the counterparts of PeBoW complex is composed of Nop7, Ytm1, and Erb1 (Miles et al., 2005;Tang et al., 2008).
Two-hybrid assays and GST pulldown experiments showed that Nop7 and Ytm1 interact directly with Erb1, but not with each other (Hölzel et al., 2005;Miles et al., 2005).In this case, we may speculate that overexpressed BOP1 may work as a dominant negative regulator of the PeBoW complex, which could explain the cell proliferation inhibitory effect of BOP1 overexpression on HEK293A cells.
In addition to our findings, how could PDGF-C recruit BOP1 is not known yet.As a secreted protein, PDGF-C activates PDGFRα, which leads to downstream signaling.Considering that BOP1 is prominently localized to the nucleolus (Strezoska et al., 2000), it is reasonable to speculate that there would be some intermediates to convey the signal from PDGFRα to BOP1.As introduced above, several signaling pathways have been identified so far to be involved in PDGF-C functions.The possible intermediate candidates may give us some hints to explore the process for PDGF-C to recruit BOP1.
In summary, this study identified a novel mechanism in which PDGF-C promotes cell proliferation partially via downregulating BOP1.
PDGF-C deficient (PDGF-C −/− ) mouse line was created and reported byDing et al. (2002).All animal experiments were performed in accordance with the National Institutes of Health guideline for the Care and Use of Laboratory animals and were approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center.Wild-type littermates were used as the control group.
Immunofluorescence stainingCells growing on glass coverslips were processed for immunofluorescence 48 h after transfection.Cells were washed with PBS briefly and then fixed in 4% paraformaldehyde for 15 min.After three times of washing with PBS, cells were pretreated in PBS supplemented with 5% Donkey serum and 0.5% Triton X-100 for 1 h.Mouse anti-BOP1 antibody (sc-390672, 1:100; Santa Cruz) in PBS supplemented with 5% Donkey serum and 0.1% Triton X-100 were incubated at 4°C overnight, and an Alexa Fluor-conjugated secondary antibody (1:500; Invitrogen) was applied for 1 h.Then the coverslips were incubated with 0.1% DAPI for 5 min.Three times of washing were applied after each antibody incubation step.Cells were observed under a confocal microscope (LSM710; Carl Zeiss).
culture system.HEK293A cells were selected for the study because they expressed very low level of endogenous PDGF-C protein in our pilot study.To ensure that protein is normally expressed and posttranslationally modified, recombinant human full-length PDGF-C (pdgf-c) plasmids were transfected into HEK293A cells, and CM was prepared.PDGF-C protein and the corresponding receptor PDGFRα were detected in the CM and cell lysate, respectively (Figure 1a,b).Thus, CM was chosen for the study of PDGF-C functions.

1
Overexpression and function of PDGF-C in HEK293A cells.HEK293A cells were transfected with plasmids encoding ZsGrn (control) or plasmids inserted with PDGF-C.CMs were collected and applied to another passage of HEK293A cells.(a) Expression of PDGF-C protein in the CM.(b) Expression of PDGFR-α in HEK293A cell lysate.(c) PDGF-C promotes HEK293A cell proliferation.The data are presented as means ± standard error of mean.**p < .01. n = 8.

F
I G U R E 2 PDGF-C downregulates BOP1 expression but does not alter its subcellular localization.HEK293A cells were cultured with CM as introduced above.(a) The expression of BOP1 determined by qPCR.The fold change relative to the level of control group is displayed.(b) The expression of BOP1 determined by Western blot.Relative expression level is displayed.(c) PDGF-C does not interfere the subcellular distribution of BOP1.HEK293A cells were treated with CM.The subcellular distribution of BOP1 was examined by immunofluorescence staining.The data are presented as means ± standard error of mean.*p < .05. n = 3 for a, and n = 5 for b.Bar = 20 μm.
Downregulation of BOP1 attenuates the mitogenic function of PDGF-C Our results demonstrate that PDGF-C downregulates the expression of BOP1, and the knockdown of BOP1 indeed promotes cell proliferation.Taken together, these results suggest a PDGF-C-BOP1 signaling to modulate cell proliferation.Then we further explore the significance of BOP1 in modulating the mitogenic functions of PDGF-C.We examined the mitogenic effects of PDGF-C CM on cells in which the expression of BOP1 was downregulated.The significance of BOP1 in modulating the mitogenic functions of PDGF-C can be viewed in two extreme ways.Hypothesis A (Figure 5a) considers BOP1 as the only effector of PDGF-C and in this case the effect of PDGF-C CM would be completely abolished by totally downregulating BOP1 expression.Hypothesis B (Figure 5b) is the other extreme, if PDGF-C promotes cell proliferation via intermediate signaling molecules other than BOP1, downregulation of BOP1 would not change the mitogenic effect of PDGF-C.To test these hypotheses, we compared the mitogenic effects of PDGF-C CM under physiological expression level of BOP1 and downregulated expression of BOP1.As shown in Figure 5c, the F I G U R E 3 pdgf-c Deficiency upregulates BOP1 expression.Skeletal muscles in the posterior limb were isolated from WT and pdgf-c deficient mice for determining the expression of BOP1.(a) The expression of BOP1 determined by qPCR.The fold change relative to the level of WT mice is displayed.(b) The expression of BOP1 determined by Western blot.All bands are cropped from the same membrane to represent the statistic results.The data are presented as means ± standard error of mean.*p < .05. n = 3 for a and n = 6 for b.F I G U R E 4 BOP1 inhibits HEK293A cell proliferation.HEK293A cell were transfected with plasmids encoding BOP1 or siBOP1.Cell proliferation was examined in full medium supplemented with serum.(a) BOP1 expression determined by Western blot.(b) Cell proliferation after the regulation of BOP1 expression.The data are presented as means ± standard error of mean.*p < .05;**p < .01;***p < .001.n = 3 for a and n = 6 for b.
Therefore, CM could better represent the overall effects of PDGF-C on cell proliferation.Furthermore, PDGF-C expressed in HEK293A cells could be fully posttranslationally modified, which is missing in bacteria expressed proteins.Nonetheless, we cannot rule out the possibility that CM contains unknown factors in addition to PDGF-C.But all our results support our hypothesis that PDGF-C modulates cell proliferation partially by a pathway involving BOP1.

F
I G U R E 5 The mitogenic effect of PDGF-C is partially regulated by BOP1.The role of BOP1 in the mitogenic effect of PDGF-C is hypothesized in two extreme ways.(a) Hypothesis A: The mitogenic effect of PDGF-C is regulated only by BOP1.(b) Hypothesis B: The mitogenic effect of PDGF-C is regulated by other factors than BOP1.(c) Downregulation of BOP1 attenuates the mitogenic effect of PDGF-C.HEK293A cells were transfected with vehicle or siBOP1.After 2 days, PDGF-C CM or the control CM were applied to those cells.The relative cell proliferation rate is displayed.The data are presented as means ± standard error of mean.*p < .05. n = 4