INTRODUCTION

Osteoarthritis (OA) is a chronic disease that is characterized by a progressive degradation of the articular cartilage that covers the surface of the synovial joints, which allow the movement of the skeleton without causing pain. Chondrocytes from patients with osteoarthritis undergo changes in the phenotype associated with an increase in catabolic and inflammatory activity¹,², along with an increase in cellular senescence and senescence-associated secretory phenotype (SASP)²,³. Our research group has previously shown that chondrocytes in the articular cartilage have long cytoplasmic projections that cross the extracellular matrix (ECM)⁴, which form connections and gap junctions (GJs) through connexin-43 channels (Cx43)⁴,⁵. In 2013, our research group published relevant results associated with alterations in the activity of Cx43 in osteoarthritis, indicating that from the disease’s early stages there is an increase and changes in the localization of the protein in the cartilage of patients with arthrosis⁶. Subsequently, using animal models, we observed that the C-terminal domain of Cx43 plays a fundamental role in the structure and composition of articular cartilage⁷.

Cx43 is involved in tissue regeneration processes in skin, heart and other tissues⁸,⁹. Several authors have reported that osteoarthritis could be included in diseases related to alterations in tissue regeneration¹⁰,¹¹. In fact, arthritic chondrocytes undergo cellular dedifferentiation and present higher levels of cell proliferation¹²,¹³, probably due to an attempt to repair the damage produced in the cartilage. The presence of chronically dedifferentiated chondrocytes triggers the progressive replacement of articular cartilage by fibrocartilage associated with degeneration and functional loss in the joint¹⁴-¹⁹. In this line of research, it is important to emphasize that the use of molecules that promote chondrogenesis, and therefore the redifferentiation of the chondrocyte, have a protective effect in OA²⁰ models. These molecules are called OA-modifying drugs (DMOADs), among which is kartogenin, which has been shown to promote chondrogenesis in human mesenchymal stem cells and also improve regeneration. of cartilage in mice subjected to inflammatory and/or mechanical damage in the joint²⁰. Other DMOADs, such as TD-198946, TAK-778 or AG-041R, have also been described as promoter molecules of chondrogenesis with therapeutic potential in repair of articular cartilage²¹-²³. The cartilage of patients with OA presents high levels of Cx43 together with alterations in the process of tissue regeneration. Our objective was to study whether alterations in Cx43 activity and intercellular communication through UCs would be related to changes in the cellular phenotype and senescence associated with disease progression.

MATERIAL AND METHODS

RESULTS

Cx43 activates the catabolic activity in chondrocytes of patients with OA

Concurring with what was observed in tissue⁶, articular chondrocytes in primary culture from donors with OA (OAc) had significantly higher Cx43 levels than those isolated from healthy donors (N) detected by flow cytometry (Figure 1A). The high levels of Cx43 were correlated with higher levels of intercellular communication through UCs, quantified by an SL/DT transfer assay of LY (Figure 1B). In order to study the effect on the cellular phenotype of high levels of Cx43 and intercellular communication through UCs, a healthy donor chondrocyte cell line, T/C-28a2, was used as a study model. Cx43 was overexpressed using a vector with the human Cx43 gene under the CMV²⁴ promoter (Figure 1C). The increase in Cx43 in the human chondrocyte cell line T/C-28a2 was correlated with an increase in the activity of the UCs detected by the SL/DT assay (Figure 1D). The gene expression assay by RT-PCR showed a significant increase in the gene expression of interleukin 1-β (IL-1β), cyclooxygenase-2 (COX-2) and metalloprotease-3 (MMP-3) when the Cx43 was overexpressed in the healthy chondrocyte line (T/C - Cx43) (Figure 1E).

Figure 1 (A) Cx43 levels analyzed by flow cytometry comparing healthy (N) and osteoarthritic (OAc) human chondrocytes in monolayer culture. n=3, mean ± standard error of the mean (EEM); ***p<0.0001; Student t test. (B) Quantification of the Scrape Loading/Dye Transfer (SL/DT) cellular communication assay comparing chondrocytes from healthy (N) and osteoarthritic (OAc) donors. n=8, mean ± SEM; **p<0.01; Mann-Whitney test. (C) On the left, immunofluorescence for Cx43 (green) in chondrocytes T/C-28a2 (T/C) and the same line transfected with a plasmid to overexpress Cx43 (T/C-Cx43). The nuclei have been stained with DAPI (blue). On the right, gene expression levels of Cx43 in these two chondrocyte lines. n=5, mean ± SEM; *p<0.05; Mann-Whitney test. (D) Quantification of the SL/DT cellular communication assay, comparing the T/28a2 (T/C) line and transfected with a plasmid to overexpress the Cx43 (T/C-Cx43). n=10, mean ± SEM; ***p<0.0001; Mann-Whitney test. (E) Levels of gene expression of IL-1β, COX-2 and MMP-3 in the T/C-28a2 line that over-expresses Cx43 (T/C-Cx43) compared to the line transfected with a control plasmid (T/C). n=4, mean ± SEM; *p<0.05; Mann-Whitney test 

Activation of cell dedifferentiation in OA

Using flow cytometric assays, we studied the levels of cell de-differentiation markers in chondrocytes from patients with osteoarthritis and chondrocytes isolated from healthy donors, in order to confirm the presence of immature chondrocytes in cartilage samples from patients with OA. By flow cytometry, higher levels of the CD166 "Stem" marker were detected in OAc in primary culture compared to healthy chondrocytes (Figure 2A). Consistent with these results, the increase of Cx43 in healthy chondrocytes (cell line) using an expression vector (T/C-Cx43 or T/C-28a2 line transfected with a plasmid to overexpress Cx43) triggered a significant increase in the levels of the two "stem-like" markers CD166 and CD105, with respect to the control cells with low levels of Cx43 (T/C-28a2) (Figure 2B).

Figure 2 (A) Measurement of CD166 dedifferentiation marker by flow cytometry in arthritic chondrocytes (OAc) and chondrocytes from healthy donors (n=9, mean ± SEM, ***p<0.0001, Mann-Whitney test). (B) Levels of markers CD105 (n=5) and CD166 (n=7) measured by flow cytometry in the T/C-28a2 cell line that over-expresses Cx43 (T/C-Cx43) compared to the same line transfected with a control plasmid (T/C). Mean ± EEM; *p<0.05; Mann-Whitney test 

The decrease in the activity of the Cx43 and the UCs activates cellular re-differentiation in OA

To reduce Cx43 activity in OAc, the effect of different molecules on the levels and activity of Cx43 was studied. In this study, we observed that the polyphenol oleuropein decreases the levels of Cx43 in OAc (Figure oleuropein decreases the levels of Cx43 in OAc (Figure 3A). The decrease in Cx43 levels improved the OA chondrocyte phenotype detected by an increase in the main marker of articular chondrocytes, collagen II (Figure 3B). The treatment of OAc with a concentration of 10 μM of oleuropein for 7 days significantly decreased levels of CD105 and CD166 dedifferentiation markers (Figure 3C), as well as the gene expression of IL-1β, IL-6, COX-2 and MMP-3 detected by flow cytometry and analysis of gene expression respectively (Figure 3D). The effect of Cx43 on cellular plasticity in OAc was confirmed in 3D culture. Modulation of Cx43 levels in the presence of 10 μM oleuropein in micromasses and in chondrogenic medium improved the structure of the extracellular matrix, detecting a significant increase in collagen II deposits and proteoglycans in the 3D structure matrix (Figure 4).

Figure 3 (A) Western blot to detect Cx43 in arthritic chondrocytes (OAc) in primary culture untreated (NT) or treated with 10 μM oleuropein (Oleu) for 2 hours. (B) Co-immunofluorescence of Cx43 (green) and collagen type II (red) of OAc treated with 10 μM oleuropein for 2 h. The cell nuclei appear in blue due to DAPI staining. The white arrows indicate Cx43 located in the cell membrane. (C) Levels of CD105 and CD166 markers measured by flow cytometry in OAc treated with 10 μM oleuropein for 7 days (n=5, mean ± SEM, **p<0.01, Mann-Whitney test). (D) Gene expression levels of IL-1β, IL-6, COX-2 and MMP-3 in OAc treated with 10 μM oleuropein for 2 hours (n=3-7, mean ± SEM; *p<0.05, ***p<0.0001, Mann-Whitney test) 

Figure 4 Sections of three-dimensional culture of arthritic chondrocytes (OAc) cultured in chondrogenic medium (MC) with/without 10 μM oleuropein for 30 days. In the upper panel, immunohistochemistry of a micromass for type II collagen (n=4-6, mean ± SEM, *p<0.05, Student's t-test). Below, staining of toluidine blue to detect proteoglycans, which produce a blue to pink-violet color shift (n=6, mean ± SEM, **p<0.01, Mann-Whitney test) 

Cx43 activates TEM and cellular senescence in OAc

The overexpression of Cx43 in the line of chondrocytes T/Cx28a2, was correlated with an increase in the nucleus of PCNA, protein used as a marker of cell proliferation, and with activation of the transcription factor related to TEM, Twist-1, detected by translocation and increased levels of the transcription factor at the nuclear level (Figure 5A). The transfected chondrocytes to overexpress the Cx43 also showed higher nuclear levels of NF-kß, one of the most important transcription factors in the regulation of synthesis of the SASP compo- nent (Figure 5A). Elevated levels of Cx43 correlated with elevated levels of factors involved in p53 cellular senescence (Figure 5B) and p16 (Figure 5C). OAc treatment with 10 μM oleuropein reduced Cx43 levels (Figure 3A) and cellular senescence detected by β-galactosidase activity by light microscopy and flow cytometry (Figure 5D). In order to confirm the effect of the Cx43 decrease in TEM and cellular senescence, the T/Cx28a2 line was transfected with a CRISPR/Cas9 plasmid, obtaining heterozygous cells for the Cx43 gene (Figure 6A). Reduced levels of Cx43 on the T/Cx28a2 line correlated with a significant decrease in the "stemlike" markers CD166 and CD105 (Figure 6B). The decrease in Cx43 levels in these cells, triggered a decrease in the levels of transcription factors Twist-1 (TEM) and NF-kβ (SASP) at the nuclear level (Figure 6C), decreasing the levels of cellular senescence , detected by β-galactosidase activity and flow cytometry (Figure 6D). T/Cx28a2 chondrocytes with low levels of Cx43 (CRISPR-Cx43) showed significantly lower levels of synthesis of the pro-inflammatory mediators IL-1β and IL-6, and of protease MMP-3, with respect to the line T/C-28a2 without transfecting.

Figure 5 (A) Western blot comparing levels of PCNA, NF-Kβ, and nuclear Twist-1 in chondrocytes that over-express Cx43 (T/C-Cx43) with respect to the same chondrocytes transfected with a control plasmid (T/C). (B) Western blot comparing total p53 levels between chondrocytes overexpressing Cx43 (T/C-Cx43) and control chondrocytes (T/C). (C) Gene expression of p16 of chondrocytes overexpressing Cx43 with respect to control cells (n=4, mean ± SEM, *p<0.05, Mann-Whitney test). (D) Above, β-galactosidase staining associated with senescence measured by X-gal rupture in arthritic chondrocytes (OAc) treated with 10 μM oleuropein for 7 days. Below, quantification by flow cytometry of β-galactosidase levels after the same treatment (n=5, mean ± SEM, *p<0.05, Mann-Whitney test) 

Figure 6 (A) On the left, gene expression levels of Cx43 in chondrocytes T/C-28a2 (T/C) and the same line with only one copy of Cx43 (CRISPR-Cx43). n=4, mean ± SEM; *p<0.05; Mann-Whitney test. On the right, immunofluorescence for Cx43 (green) in chondrocytes T/C-28a2 (T/C) and the same line transfected with only one copy of Cx43 (CRISPR-Cx43). The nuclei have been stained with DAPI (blue). (B) Levels of the CD105 and CD166 markers measured by flow cytometry in the T/C-28a2 cell line with a single copy of Cx43 (CRISPR-Cx43) compared to the same line without transfection (T/C). n=7, mean ± SEM; *p<0.05, **p<0.01; Mann-Whitney test (C) Western blot to detect Twist-1, NFkβ and N-Cadherin comparing a nuclear extract of the T/C-28a2 (T/C) line and from the same line with low amount of Cx43 (CRISPR-Cx43). Lamina protein A has been used as a load control. (D) Quantification by flow cytometry of β-galactosidase levels in the T/C-28a2 line (T/C) and the same line with only one copy of Cx43 (CRISPR-Cx43). n=4, mean ± SEM; *p<0.05; Mann-Whitney test. (E) Gene expression levels of IL-1β, IL-6 and MMP-3 measured in the same cells. n=4, mean ± SEM; *p<0.05; Mann-Whitney test) 

DISCUSSION

During osteoarthritis, the chondrocytes have increased levels of the transmembrane protein Cx43⁶ and their phenotype is altered preventing them from participating in tissue regeneration and carrying out their function, triggering progressive tissue degeneration. The dedifferentiation related to epithelial-mesenchymal transition phenomena (TEM) is a cellular process that participates in the regeneration of tissues by allowing the cells to dedifferentiate into a more immature state to activate processes, including cell proliferation and migration, with the objective of replacing the damaged cells and remodeling the extracellular matrix²⁵,²⁶. However, when this dedifferentiation occurs chronically it can cause the development of fibrosis in the context of tissue regeneration²⁷,²⁸. In this study we have described that the levels of Cx43 and intercellular communication through UCs in osteoarthritis correlate positively with the cell de-differentiation markers CD105 and CD166. In addition, we have verified that this state can be partially reversed by the use of molecules that decrease the levels of Cx43, improving the phenotype of arthritic chondrocytes and

MEC in in vitro tests. The decrease in Cx43 gave rise to cellular re-differentiation and, therefore, to a lower expression of pro-inflammatory cytokines and degrading enzymes of the articular cartilage matrix. Our results also show that high levels of Cx43 in chondrocytes are related to an increase in senescence associated with a higher expression of p16^INK4a and high levels of p53. Recent studies highlight the importance of senescence in osteoarthritis²⁹-³³. In fact, Jeon et al. published an article in Nature Medicine where they showed senescence as a new therapeutic target to treat osteoarthritis and promote the regeneration of cartilage³³,³⁴. In this study we demonstrate for the first time the relationship between the over activity of Cx43 in human chondrocytes and the activation of dedifferentiation and cellular senescence that lead to alterations in the regeneration process and favor the progress of the disease. From these results, therapies aimed at decreasing Cx43 levels in osteoarthritis arise as an interesting therapeutic approach for osteoarthritis.

In conclusion, these findings suggest that the increase in Cx43 activity reached from very early stages of OA⁶ could contribute to the degeneration of articular cartilage and joint by activating cellular dedifferentiation via TEM and cellular senescence, contributing to the synthesis of enzymes that degrade the release of cytokines that contribute to the degenerative process in the joint. These results demonstrate that Cx43 and UCs act as a regulator of dedifferentiation/re-differentiation and senescence in chondrocytes, probably activating proteins related to TEM, such as Twist-1, and pro-inflammatory cytokines such as IL-1β. The decrease in Cx43 levels in OAc promotes its re-differentiation, decreasing the expression of inflammatory mediators and senescence, and in turn is accompanied by a greater deposition of Col2A1 and proteoglycans in the extracellular matrix. The use of molecules such as oleuropein and the design of studies to decrease the activity of Cx43 in vivo is probably a first step in the development of innovative therapeutic strategies for the effective treatment of osteoarthritis from early stages of the disease by restoring tissue regeneration.