Osteoarthritis:Mechanistic Insights, Senescence, and Novel Therapeutic Opportunities

Osteoarthritis (OA) is the most common joint disease. In the last years, the research community has focused on understanding the molecular mechanisms that led to the pathogenesis of the disease, trying to identify different molecular and clinical phenotypes along with the discovery of new therapeutic opportunities. Different types of cell-to-cell communication mechanisms have been proposed to contribute to OA progression, including mechanisms mediated by connexin43 (Cx43) channels or by small extracellular vesicles. Furthermore, changes in the chondrocyte phenotype such as cellular senescence have been proposed as new contributors of the OA progression, changing the paradigm of the disease. The use of different drugs able to restore chondrocyte phenotype, to reduce cellular senescence and senescence-associated secretory phenotype components, and to modulate ion channel activity or Cx43 appears to be promising therapeutic strategies for the different types of OA. In this review, we aim to summarize the current knowledge in OA phenotypes related with aging and tissue damage and the new therapeutic opportunities currently available

Recruitment of RNA molecules by connexin RNA-binding motifs: Implication in RNA and DNA transport through microvesicles and exosomes

Connexins (Cxs) are integral membrane proteins that form high-conductance plasma membrane channels, allowing communication from cell to cell (via gap junctions) and from cells to the extracellular environment (via hemichannels). Initially described for their role in joining excitable cells (nerve and muscle), gap junctions (GJs) are found between virtually all cells in solid tissues and are essential for functional coordination by enabling the direct transfer of small signalling molecules, metabolites, ions, and electrical signals from cell to cell. Several studies have revealed diverse channel-independent functions of Cxs, which include the control of cell growth and tumourigenicity. Connexin43 (Cx43) is the most widespread Cx in the human body. The myriad roles of Cx43 and its implication in the development of disorders such as cancer, inflammation, osteoarthritis and Alzheimer's disease have given rise to many novel questions. Several RNA- and DNA-binding motifs were predicted in the Cx43 and Cx26 sequences using different computational methods. This review provides insights into new, ground-breaking functions of Cxs, highlighting important areas for future work such as transfer of genetic information through extracellular vesicles. We discuss the implication of potential RNA- and DNA-binding domains in the Cx43 and Cx26 sequences in the cellular communication and control of signalling pathwaysThis work was supported in part through funding from the Society for Research on Bone and Mineral Metabolism - Grant number FEIOMM2016 (to M.D.M.), by grant PRECIPITA-2015-000139 from the FECYT-Ministry of Economy and Competitiveness (to M.D.M), by grants PI13/00591 and PI16/00035 from the Health Institute “Carlos III” (ISCIII, Spain) and co-financed by the European Regional Development Fund, “A way of making Europe” from the European Union (to M.D.M.), by a grant from Xunta de Galicia (pre-doctoral fellowship) to M.V.-E., and by a grant from the Ministry of Education, Culture and Sports, Spain (FPU grant to M.R.-C.M.)S

Senolytic Activity of Small Molecular Polyphenols from Olive Restores Chondrocyte Redifferentiation and Promotes a Pro-Regenerative Environment in Osteoarthritis

[Abstract] Articular cartilage and synovial tissue from patients with osteoarthritis (OA) show an overactivity of connexin43 (Cx43) and accumulation of senescent cells associated with disrupted tissue regeneration and disease progression. The aim of this study was to determine the effect of oleuropein on Cx43 and cellular senescence for tissue engineering and regenerative medicine strategies for OA treatment. Oleuropein regulates Cx43 promoter activity and enhances the propensity of hMSCs to differentiate into chondrocytes and bone cells, reducing adipogenesis. This small molecule reduce Cx43 levels and decrease Twist-1 activity in osteoarthritic chondrocytes (OACs), leading to redifferentiation, restoring the synthesis of cartilage ECM components (Col2A1 and proteoglycans), and reducing the inflammatory and catabolic factors mediated by NF-kB (IL-1ß, IL-6, COX-2 and MMP-3), in addition to lowering cellular senescence in OACs, synovial and bone cells. Our in vitro results demonstrate the use of olive-derived polyphenols, such as oleuropein, as potentially effective therapeutic agents to improve chondrogenesis of hMSCs, to induce chondrocyte re-differentiation in OACs and clearing out senescent cells in joint tissues in order to prevent or stop the progression of the disease.Xunta de Galicia; IN607B 2017/21Xunta de Galicia; ED481A-2015/188Xunta de Galicia; IN606B-2019/004Xunta de Galicia; IN606B-2017/014This work was supported in part through funding from the Spanish Foundation for Research on Bone and Mineral Metabolism (FEIOMM), grant PRECIPITA-2015-000139 from the FECYT-Ministry of Economy and Competitiveness (to M.D.M.), grant PI16/00035 and PI19/00145 from the Health Institute ‘Carlos III’ (ISCIII, Spain), the European Regional Development Fund, ‘A way of making Europe’ from the European Union (to M.D.M.) and a grant from Xunta de Galicia IN607B 2017/21 (to M.D.M.). M.V.-E. was funded with a predoctoral (ED481A-2015/188) and a postdoctoral fellowship (IN606B-2019/004) from Xunta de Galicia. P.C.-F. was funded with a postdoctoral fellowship (IN606B-2017/014) from Xunta de Galicia

Cdc14 phosphatase promotes segregation of telomeres through repression of RNA polymerase II transcription

Kinases and phosphatases regulate messenger RNA synthesis through post-translational modification of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (ref. 1). In yeast, the phosphatase Cdc14 is required for mitotic exit2,3 and for segregation of repetitive regions4. Cdc14 is also a subunit of the silencing complex RENT (refs 5, 6), but no roles in transcriptional repression have been described. Here we report that inactivation of Cdc14 causes silencing defects at the intergenic spacer sequences of ribosomal genes during interphase and at Y′ repeats in subtelomeric regions during mitosis. We show that the role of Cdc14 in silencing is independent of the RENT deacetylase subunit Sir2. Instead, Cdc14 acts directly on RNA polymerase II by targeting CTD phosphorylation at Ser 2 and Ser 5. We also find that the role of Cdc14 as a CTD phosphatase is conserved in humans. Finally, telomere segregation defects in cdc14 mutants4 correlate with the presence of subtelomeric Y′ elements and can be rescued by transcriptional inhibition of RNA polymerase II

RNAP-II Molecules Participate in the Anchoring of the ORC to rDNA Replication Origins

<div><p>The replication of genomic DNA is limited to a single round per cell cycle. The first component, which recognises and remains bound to origins from recognition until activation and replication elongation, is the origin recognition complex. How origin recognition complex (ORC) proteins remain associated with chromatin throughout the cell cycle is not yet completely understood. Several genome-wide studies have undoubtedly demonstrated that RNA polymerase II (RNAP-II) binding sites overlap with replication origins and with the binding sites of the replication components. RNAP-II is no longer merely associated with transcription elongation. Several reports have demonstrated that RNAP-II molecules affect chromatin structure, transcription, mRNA processing, recombination and DNA repair, among others. Most of these activities have been reported to directly depend on the interaction of proteins with the C-terminal domain (CTD) of RNAP-II. Two-dimensional gels results and ChIP analysis presented herein suggest that stalled RNAP-II molecules bound to the rDNA chromatin participate in the anchoring of ORC proteins to origins during the G1 and S-phases. The results show that in the absence of RNAP-II, Orc1p, Orc2p and Cdc6p do not bind to origins. Moreover, co-immunoprecipitation experiments suggest that Ser2P-CTD and hypophosphorylated RNAP-II interact with Orc1p. In the context of rDNA, cryptic transcription by RNAP-II did not negatively interfere with DNA replication. However, the results indicate that RNAP-II is not necessary to maintain the binding of ORCs to the origins during metaphase. These findings highlight for the first time the potential importance of stalled RNAP-II in the regulation of DNA replication.</p> </div

The inhibition of transcription by RNAP-II does not affect the binding of replication components to the rDNA locus.

<p>(<b>a</b>) Chromatin immunoprecipitation (ChIP) analysis of RNAP-II (4H8), Orc1p, Orc2p and Cdc6p binding within the intergenic spacers (primers 15 to 22), <i>35S</i> (primer 13 and 23) and <i>5S</i> gene regions (primer 18) in wild type cells. Orc1p, Orc2p and Cdc6p bound to cohesin (CAR, primer 19 and 20) and ARSs (p20, p21). (<b>b</b>) ChIP analysis of Orc1p, Orc2p and Cdc6p bound to the ARS in a <i>rpb1–1 ts</i> strain growing at 25°C. The results were obtained for untreated and cells that were treated with AM for 1 hour or DRB for 4 hours. All values are expressed as the mean ± S.E.M. n = 2 or n = 3. **p<0.005, *p<0.05 for the Student’s t<i>-</i>test, untreated versus treated. (<b>c</b>) ChIP analysis of the <i>rpb1–1 ts</i> strain growing at 25°C or shifted at 37°C for 30 minutes. Mean ± S.E.M. n = 3 or n = 4. **p<0.005, *p<0.05 for Student’s t<i>-</i>test, 25°C versus 37°C. A sequence located in chromosome VI was used as a negative control (background) for the binding of the replication proteins.</p

RNAP-II participates in the anchoring of Orc1p, Orc2p and Cdc6p to the ARS.

<p>(<b>a</b>) 2D gel of the RIs corresponding to an asynchronous culture (<i>rpb1–1</i> strain) that was arrested with α-factor in G1 at 25°C and subsequently was divided into four aliquots. α-factor at 25°C and α-factor at 37°C for 45 additional minutes: Two flasks were released with pronase, and the cells were grown at 25°C or shifted to 37°C for 45 minutes. (<b>b</b>) ChIP analysis of Orc1p, Orc2p and Cdc6p within the IGS regions in <i>rpb1–1</i> cells arrested in G1 at 25°C and shifted to 37°C. On the right is the ChIP analysis obtained after releasing cells 45 minutes after pronase at 25°C or 37°C. (<b>c</b>) and (<b>d</b>) ChIP analysis and images corresponding to a 2D gel obtained from an asynchronous culture (wild type) that was arrested in G1 with α-factor and subsequently divided into four aliquots as described above. (<b>e</b>) and (<b>f</b>) ChIP analysis and 2D gels show the RIs of the asynchronous culture (wild type) used to arrest cells in G1 (α-factor). The cells were incubated for 45 additional minutes in the presence or absence of 10 µg/ml of AM. The cells that were released from α-factor were incubated in the presence or absence of AM. The fold enrichment relative to a sequence located in ChVI is shown. Mean ± S.E.M. n = 2 or n = 3. **p<0.005, *p<0.05 for Student’s t<i>-</i>test, 25°C versus 37°C, untreated versus treated.</p

The replication of rDNA is affected in the absence of the largest subunit of RNAP-II.

<p>(<b>a</b>) Diagram of the rDNA with the locations of the replication barrier (RFB), replication origin (ARS) and cohesin binding sequences (CAR). Below, the theoretical schemes for the two-dimensional agarose gel electrophoresis of chromatin digested with <i>Bgl</i>II are depicted. The accumulation of RIs at the RFB is expected at 1.49X. 1X represents the position of the linear, unreplicated fragment. The hybridisation probe used for the Southern protocol and hybridisation was amplified by PCR and column purified (Qiagen). The amplified fragment is represented above<b>.</b> The direction of the anticlockwise and clockwise replication fork is shown below. (<b>b</b>) Results obtained after the isolation of DNA from asynchronous cultures growing exponentially at different temperatures. Higher exposure images are shown below. Significantly fewer amounts of RIs were detected in the absence of <i>RPB1</i> (<i>rpb1–1 ts</i> strain). 1X signals were similar among the 4 experiments (25°C, 37°C for 30 minutes, 37°C for 1 hour and 37°C for 3 hours). The same number of cells was analysed. (<b>c</b>) Results obtained after <i>fob1</i> deletion in the <i>rpb1–1 ts</i> strain. (<b>d</b>) To control the effect of the temperature on DNA replication, a wild type strain (BY4741) was used. (<b>e</b>) Results obtained after the isolation of DNA from a <i>fob1</i>Δ strain containing approximately 190 or 25 rDNA copies. A higher exposure image is shown on the right for the 25 copies strain.</p