Pathophysiology of connexin-related disorders

Connexins are integral membrane proteins that form plasma membrane channels, allowing cell-matrix and cell to cell communication. Initially described in joining excitable cells (nerve and muscle), gap junctions (GJs) are found joining virtually all cells in solid tissues and are essential for the functional co-ordination of organs by enabling direct transfer of small signalling molecules, metabolites, ions, and electrical signals.

Several studies have revealed diverse channel-independent functions of Cxs, including control of cell growth and tumorigenicity. In fact some reports have demonstrated that Cx26 and Cx43 can act as a tumor suppressor in melanoma and breast cancer tumors. However, the role of connexins in mediating metastasis remains controversial. The myriad roles of Cx43 and its implication in the development of diseases such as cancer, osteoarthritis or psoriasis have rise to many novel questions.

Study of the functions of Cx43 and gap junctions in articular cartilage

We have found that connexins are altered in the cartilage of OA patients, with Cx43 being overrepresented in the damaged zones and a loss of Cx43 membrane localisation in non-damaged areas (Mayan et al., 2013). OA disease is characterised by multiple molecular alterations and most of the changes that occur in OA cartilage have previously been described for connexin channels in other connexin-related diseases. These results encouraged us to investigate the functions of Cx43 in normal cartilage and in cartilage from patients with OA, searching for some alteration that could explain the degeneration of the ECM observed in patients with OA. The goal of this project is to find effective therapies for arthritis

Osteoarthritis (OA), together with heart diseases and cancer is a leading cause of short-term disability in the western world, manifesting as a significant health expenditure, decreased quality of life and disability in older people. Despite the impact of the great number of publications and research groups studying arthritis, the primary cause of the disease has not yet been described. Our research group is investigating the molecular mechanism that is most  likely altered in patients with osteoarthritis.

Articular cartilage, which allows the painless, low-friction movement of the joints, is a tissue composed of an extracellular matrix (ECM) formed mainly of type II collagen and proteoglycans that cover the ends of bones. Chondrocytes are highly specialised cells embedded in the ECM that provide the articular cartilage with remarkable mechanical properties. The chondrocytes are responsible for the formation and remodelling of the tissue throughout adult life. Chondrocytes embedded in the articular cartilage are rounded cells located in small cavities called lacuna. Usually, there is only one cell per lacunae. It was believed that chondrocytes did not directly interact with each other except through the diffusion of substances into the matrix. However, proper cell-to-cell communication is essential to maintain the homeostasis of any tissue by enabling the cells to timely and uniformly respond to a localised stimuli or damage. Together, the lack of blood vessels and the isolation of the cells, suggest that adult cartilage hardly possess any regenerative capacity. However, this is most likely not the case with articular cartilage, as aging reduces the cell density and the thickness of cartilage, but it is still able to maintain its structure and function. On the other hand, the progressive degeneration of the cartilage matrix leads to osteoarthritis, which is a degenerative joint disease characterised by the degeneration and loss of the articular cartilage structure and function. Until now, pain management and surgery have been the only available treatments for osteoarthritis.

Anatomical, histological and physiological studies have assumed that chondrocytes are metabolically active cells isolated in their own lacunae. However, results from our group have revealed, for the first time, that chondrocytes in tissue have at least two long cytoplasmic extensions, which spread out thinly (200 nm in width) along the ECM and physically connect cells located in different lacuna (Mayan et al., 2013). In another report, we published that human articular chondrocytes contain high levels of integral membrane proteins called connexins (Mayan et al., 2013), which are responsible for electrical coupling between cells, such as the electrical synapses in neurons or the coordinated depolarisation of cardiac muscles. In effect, our results demonstrated that primary chondrocytes have the capacity to form functional voltage-dependent gap junction (GJ) channels (Mayan et al., 2013). These results were very well received at the 2011 International Gap Junction Conference, the 2012 Osteoarthritis Research Society International meeting (OARSI), the 2012 and 2013 ACR meetings and the 2013 OARSI meeting (Dr. Maria D. Mayan gave two talks at two different sections).

We have found that connexins are altered in the cartilage of OA patients, with Cx43 being overrepresented in the damaged zones and a loss of Cx43 membrane localisation in non-damaged areas (Mayan et al., 2013). OA disease is characterised by multiple molecular alterations and most of the changes that occur in OA cartilage have previously been described for connexin channels in other connexin-related diseases. These results encouraged us to investigate the functions of Cx43 in normal cartilage and in cartilage from patients with OA, searching for some alteration that could explain the degeneration of the ECM observed in patients with OA. The goal of this project is to find effective therapies for arthritis [More].

Podoplanin, MASL and arthritis

PDPN is expressed at low levels in osteocytes, osteoblasts, chondrocytes, and synovial cells. However, PDPN expression is drastically increased in synovial tissue and Th17 cells in patients with RA. Furthermore, immunohistochemistry experiments performed by our group revealed that the cartilage explants from patients with osteoarthritis contain higher levels of PDPN expression than healthy cartilage.

Podoplanin (PDPN) is a type-1 transmembrane glycoprotein containing an extracellular domain highly O-glycosylated with α-2,3-sialic acid linked to galactose. PDPN is involved in cell migration and can regulate the inflammatory reactions produced by neutrophils and macrophages. In addition, inflammatory factors including IFN-γ, TGF-ß, TNF-α, and IL-1ß, which cause the degradation of cartilage and the synovial joint, can increase PDPN expression. Indeed, anti-TNF therapy has been shown to significantly reduce PDPN expression in synovial cells in rheumatoid arthritis (RA) patients.

PDPN is expressed at low levels in osteocytes, osteoblasts, chondrocytes, and synovial cells. However, PDPN expression is drastically increased in synovial tissue and Th17 cells in patients with RA. Furthermore, immunohistochemistry experiments performed by our group revealed that the cartilage explants from patients with osteoarthritis contain higher levels of PDPN expression than healthy cartilage. Moreover, the treatment of chondrocytes with a lectin (MASL)-targeting PDPN, protects chondrocytes from reactive oxygen species (ROS) and inflammatory cytokines at concentrations that do not affect cell viability, adhesion, or growth. These results led to the development of a patent [More]. We are further investigating the role of PDPN in the inflammatory response to prevent joint damage.

Transcriptional regulation of the frataxin gene: identifying novel therapeutic targets for Friedreich’s ataxia

Maria Mayan during her first postdoctoral position, together with Nadine Rothe and Richard Festenstein and in collaboration with Ana Pombo, found that a Ser2-phosphorylated form of RNAPII stalled at a CpG-rich region near the FXN promoter is rapidly degraded in the presence of the GAA-repeat expansion but not degraded in normal cells.

Friedreich’s ataxia (FRDA), the most frequently inherited progressive ataxia, is caused by a GAA-repeat expansion in the first intron of the FXN gene. This expansion leads to the repression of FXN and a partial deficiency in the mitochondrial Frataxin protein. Maria Mayan during her first postdoctoral position, together with Nadine Rothe and Richard Festenstein and in collaboration with Ana Pombo, found that a Ser2-phosphorylated form of RNAPII stalled at a CpG-rich region near the FXN promoter is rapidly degraded in the presence of the GAA-repeat expansion but not degraded in normal cells. These results revealed key aspects of the dynamics of RNAPII activity bound to the FXN gene identifying novel potential therapeutic avenues for Friedreich’s ataxia, and other diseases with similar underlying mechanisms. These results were further developed by other co-authors and are pending publication.