Suramin enhances chondrogenic properties by regulating the p67phox/PI3K/AKT/SOX9 signalling pathway

Abstract

Aims: Autologous chondrocyte implantation (ACI) is a promising treatment for articular cartilage degeneration and injury; however, it requires a large number of human hyaline chondrocytes, which often undergo dedifferentiation during in vitro expansion. This study aimed to investigate the effect of suramin on chondrocyte differentiation and its underlying mechanism.

Methods: Porcine chondrocytes were treated with vehicle or various doses of suramin. The expression of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN); COL1A1; COL10A1; SRY-box transcription factor 9 (SOX9); nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX); interleukin (IL)-1β; tumour necrosis factor alpha (TNFα); IL-8; and matrix metallopeptidase 13 (MMP-13) in chondrocytes at both messenger RNA (mRNA) and protein levels was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot. In addition, the supplementation of suramin to redifferentiation medium for the culture of expanded chondrocytes in 3D pellets was evaluated. Glycosaminoglycan (GAG) and collagen production were evaluated by biochemical analyses and immunofluorescence, as well as by immunohistochemistry. The expression of reactive oxygen species (ROS) and NOX activity were assessed by luciferase reporter gene assay, immunofluorescence analysis, and flow cytometry. Mutagenesis analysis, Alcian blue staining, reverse transcriptase polymerase chain reaction (RT-PCR), and western blot assay were used to determine whether p67^phox was involved in suramin-enhanced chondrocyte phenotype maintenance.

Results: Suramin enhanced the COL2A1 and ACAN expression and lowered COL1A1 synthesis. Also, in 3D pellet culture GAG and COL2A1 production was significantly higher in pellets consisting of chondrocytes expanded with suramin compared to controls. Surprisingly, suramin also increased ROS generation, which is largely caused by enhanced NOX (p67^phox) activity and membrane translocation. Overexpression of p67^phox but not p67^phoxAD (deleting amino acid (a.a) 199 to 212) mutant, which does not support ROS production in chondrocytes, significantly enhanced chondrocyte phenotype maintenance, SOX9 expression, and AKT (S473) phosphorylation. Knockdown of p67^phox with its specific short hairpin (sh) RNA (shRNA) abolished the suramin-induced effects. Moreover, when these cells were treated with the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) inhibitor LY294002 or shRNA of AKT1, p67^phox-induced COL2A1 and ACAN expression was significantly inhibited.

Conclusion: Suramin could redifferentiate dedifferentiated chondrocytes dependent on p67^phox activation, which is mediated by the PI3K/AKT/SOX9 signalling pathway.Cite this article: Bone Joint Res 2022;11(10):693-708.

Keywords: Alcian blue; Chondrocyte redifferentiation; Col2a1; NADPH oxidase; RNA; Reactive oxygen species; Suramin; chondrocytes; collagen; mRNA; p67phox; phosphate; reverse transcriptase-polymerase chain reaction; staining; western blot.

Fig. 1

Effect of suramin on chondrogenic and hypertrophic marker gene expression in chondrocytes. The P3 passage of chondrocytes were treated: a) with or without 10 μM suramin for various timepoints; or b) with differents amount of suramin (0 to 10 μM) for one day, and the protein expressions of collagen, type II, alpha 1 (COL2A1), COL10A1, and aggrecan (ACAN) were analyzed by western blot, with β-actin as the loading control. c) The chondrocytes were incubated with or without 10 μM suramin for 0.5 hours to 24 hours, and the messenger RNA (mRNA) levels of Col2a1 and Acan were detected by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). d) Early (P1) or later passage (P6) of chondrocytes were treated with or without suramin, and the protein expressions of COL2A1, COL10A1, and ACAN were analyzed by western blot. e) Relative mRNA levels in chondrocytes treated with or without suramin were tested for cartilage-specific markers such as Col2a1, Col10a1, and Acan, as well as Col1 (dedifferentiation marker) using qRT-PCR (n = 3). Values were normalized to glyceraldehyde 3-phosphate dehydrogenase levels. Data are shown as mean (standard deviation (SD)); *p < 0.05, **p < 0.01, and ***p < 0.001. f) Representative immunofluorescence images of COL2A1 (green) and COL10a1 (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). g) Chondrocyte pellets were treated with or without suramin for 28 days and dimethylmethylene blue (DMMB) assay determining total sulphated glycosaminoglycan content normalized to DNA content; **p < 0.01 between groups. Data shown are representative of three independent replicates. h) Alcian blue staining was used to examine the effects of suramin on GAG expression in pellet culture of chondrocytes. Scale bar = 50 μm. i) Immunofluorescence and immunohistochemistry for COL2A1 and COL1A1 on chondrocyte pellets in the presence or absence of 10 μM suramin for 28 days.

Fig. 2

Involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) in the suramin-induced reactive oxygen species (ROS) production. Cells were treated with 0 to 10 μM suramin for various time periods as indicated. Then, the cells were further incubated with 100 μM dichloro-dihydro-fluorescein diacetate (DCFH-DA) for 30 minutes before detection of the fluorescence by fluorescence microscopy (magnification: 100×) (a, b, left panel) or by flow cytometry (d, left panel). c) Cells were pretreated with N-acetyl-L-cysteine (NAC) (c), rotenone (c), and diphenyleneiodonium chloride (DPI) (c, d) or vehicle, followed by treatment with 10 μM suramin for 30 minutes. Then, the cells were further incubated with 100 μM DCFH-DA for 30 minutes before detection of the fluorescence by fluorescence microscopy or by flow cytometry (a to d, f). Results are expressed as mean values; ^###p < 0.001 compared to vehicle control and ***p < 0.001, **p < 0.01, *p < 0.05 compared to suramin treatment alone. e) Cells were treated with 10 μM suramin for various time periods as indicated to measure their NOX activity, as described in the Methods section. f) Bar diagram showing quantitative data of 2',7'-dichlorofluorescein-positive cells.

Fig. 3

Differential expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) during suramin-induced differentiation of primary chondrocytes. a) and c) The chondrocytes were treated with 10 μM suramin for 16 hours. Then we measured Nox1, Nox2, Nox4, p47, p67, and glyceraldehyde 3-phosphate dehydrogenase (Gapdh) messenger RNA (mRNA) levels using reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR). b) and d) Total cell lysates were analyzed by western blot with specific antibodies as indicated. e) pEGFP-p67 plasmid was transciently transfected into chondrocytes and incubated with or without 10 mM of suramin. Thereafter, the cells were fixed with formaldehyde and permeabilized with Triton X-100. The images were observed with an immunofluoresence microscope through a 40× objective. f) Cells were treated with 10 μM suramin for 30 and 60 minutes. The cytoplasmic and membrane protein fractionations were collected as described in the Methods section to detect p67 ^phox expression by western blot. g) and h) The chondrocytes were transfected with NOX2 or p67^phox luciferase plasmid, respectively, and then incubated with 10 μM suramin for different times as indicated. The luciferase activity was determined and normalized with the amount of total protein. Values are means and standard deviations of triplicate measurements. *p < 0.05, **p < 0.01 compared with untreated control. C, control.

Fig. 4

Suramin positively regulated NOX2/p67^phox expression and was involved in differentiation of primary chondrocytes. a) to c) The chondrocytes were pretreated with a) N-acetyl-L-cysteine (NAC), b) diphenyleneiodonium chloride (DPI), and c) apocynin for one hour followed by treatment with or without 10 μM suramin for 24 hours. Total cell lysates were analyzed by western blot with specific antibodies as indicated. d) Chondrocytes were transfected with pLKO.1-p67 ^phox -shRNA or empty vector followed by treatment with 10 μM suramin for 24 hours, then total cell lysates were collected and subjected to western blot with specific antibodies as indicated. **p < 0.01, ***p < 0.001 compared with untreated control; ###p < 0.001 compared to the control. ACAN, aggrecan; COL2A1, collagen, type II, alpha 1; DPI, diphenyleneiodonium chloride; NOX, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase; shRNA, short hairpin (sh) RNA.

Fig. 5

Differentiation of primary chondrocytes in depleted and overexpressed p67^phox. a) Later passage of chondrocytes were transiently transfected with various doses of p67^phox (pcDNA- p67^phox) or control (pcDNA3.1) plasmid for additional 24 hours, to detect the messenger RNA (mRNA) and protein expression of chondrogenic and differentiation marker as indicated by using reverse transcriptase polymerase chain reaction (RT-PCR) or western blot. b) Early stage of chondrocytes were transfected with specific shRNA of p67^phox (pLKO.1-p67^phox-shRNA) or a luciferase control (pLKO.1-shLuc) to measure mRNA and protein expression of chondrogenic and differentiation marker as indicated by using RT-PCR or western blot. The expression of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) or β-ACTIN was used as an internal control of RT-PCR or western blot, respectively. c) Late passage of primary chondrocytes were treated as in Figure 5a, and senescence-associated β-galactosidase (SA-β-gal) activity and Alcian Blue staining (left panel) were used to detect these. Representative photomicrograph of the SA-β-gal assay is shown. The percentage of β-galactosidase-positive cells in each group was compared and is illustrated in the histogram; n = 3 in each experiment. d) Early passage of chondrocyte were treated as in Figure 5b, and senescence-associated β-galactosidase (SA-β-gal) activity and Alcian Blue staining (magnification: 100×) (left panel) were used to detect these. Representative photomicrograph of the SA-β-gal assay is shown. The percentage of β-galactosidase-positive cells in each group was compared and is illustrated in the histogram; n = 3 in each experiment. e) and f) The chondrocytes were transiently transfected with p67^phox (pcDNA-p67^phox), pcp67△ AD (a.a 199 to 212), or control (pcDNA3.1) plasmid, to detect the mRNA and protein expression of COL2A1 and aggrecan as indicated by using RT-PCR or western blot. *p < 0.05, **p < 0.01, ***p < 0.001 compared with untreated control. shRNA, short hairpin (sh) RNA.

Fig. 6

Effect on dedifferentiation marker expression in depleted and overexpressed p67^phox. a) and b) The chondrocytes were transiently transfected with various doses of p67^phox plasmid (pcDNA-p67^phox) or control (pcDNA3.1) to detect messenger RNA (mRNA) and protein expression of dedifferentiation marker as indicated by using reverse transcriptase polymerase chain reaction (RT-PCR) or western blot. c) and d) The chondrocytes were transfected with specific shRNA of p67^phox (pLKO.1-p67^phox-shRNA) or a luciferase control (pLKO.1-shLuc) to measure mRNA and protein expression of dedifferentiation marker as indicated by using RT-PCR or western blot. The expression of glyceraldehyde 3-phosphate dehydrogenase (gapdh) or β-actin was used as an internal control of RT-PCR or western blot, respectively. Results are expressed as mean value; ***p < 0.001, **p < 0.01, *p < 0.05 compared to transfected with the pcDNA or shLuc plasmid. IL, interleukin; MMP, matrix metallopeptidase; shRNA, short hairpin (sh) RNA; TNF, tumour necrosis factor.

Fig. 7

Phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway was involved in p67^phox-induced chondrocyte differentiation. a) The relationship between the ERK and PI3K/AKT pathway and p67^phox was also measured in chondrocytes by western blot analysis. β-actin served as protein loading control. *p < 0.05, **p < 0.01, ***p < 0.001 compared to transfected pcDNA alone. b) Later passage of chondrocytes were transiently transfected with various doses of p67^phox plasmid (pcDNA-p67^phox) or control (pcDNA3.1). p67^phox-overexpressed cells were pretreated with either 5 or 10 μmol/l of LY294002 for three hours, and then the expression of collagen, type II, alpha 1 (COL2A1) and aggrecan (ACAN) protein was measured by western blot analysis. Each assay was performed in triplicate, and the results shown as means (standard deviations (SDs)) of four independent experiments (p < 0.001). c) The pcp67^phox plasmids (1 μg) were used to cotransfect chondrocytes with pLKO.1-AKT1-shRNA (1 or 2 μg) for 24 hours, and the cell lysates were harvested to determine p67, p-AKT^S473, COL2A1, ACAN, and β-ACTIN expression. ###p < 0.001, ##p < 0.01 compared to transfected pcDNA alone; *p < 0.05, **p < 0.01, ***p < 0.001 compared to transfected with wild type of pcp67 plasmid. shRNA, short hairpin (sh) RNA.

Fig. 8

p67^phox-induced chondrocyte differentiation was dependent on PI3K/AKT/SOX9 pathways. a) The chondrocytes were transiently transfected with various doses of p67^phox plasmid (pcDNA-p67^phox) or control (pcDNA3.1), and then the expression of p67^phox and SOX9 protein was measured by western blot analysis. b) and c) The chondrocytes were transiently transfected with various doses of p67^phox plasmid (pcDNA-p67^phox) or control (pcDNA3.1). p67^phox-overexpressed cells were pretreated with either 5 or 10 μmol/l of LY294002 for three hours, and then the protein and messenger RNA (mRNA) expression of SOX9 was measured by western blot and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. ###p < 0.001 compared with transfected pcDNA alone; ***p < 0.001 compared with untreated control. AKT, protein kinase B; PI3K, phosphoinositide 3-kinase; SOX9, SRY-box transcription factor 9.

Fig. 9

Scheme illustrating the possible mechanism of suramin-regulated cellular differentiation of chondrocytes. AKT, protein kinase B; COL2A1, collagen, type II, alpha 1; ROS, reactive oxygen species; SOX9, SRY-box transcription factor 9.