Thrombospondin-4 promotes bladder cancer cell migration and invasion via MMP2 production.

Bladder cancer (BC) is the second most common urological tumour in Western countries. Approximately, 80% of patients with BC will present with non-muscle invasive bladder cancer (NMIBC), whereas a quarter will have muscle invasive disease (MIBC) at the time of BC diagnosis. However, patients with NMIBC are at risk of BC recurrence or progression into MIBC, and an MIBC prognosis is determined by the presence of progression and metastasis. Matrix metalloproteinase 2 (MMP2), a type of matrix metalloproteinase (MMP), plays a major role in tumour invasion and is well-characterized in BC prognosis. In BC, the mechanisms regulating MMP2 expression, and, in turn, promote cancer invasion, have hardly been explored. Thrombospondin-4 (THBS4/TSP4) is a matricellular glycoprotein that regulates multiple biological functions, including proliferation, angiogenesis, cell adhesion and extracellular matrix modelling. Based on the results of a meta-analysis in the Gene Expression Profiling Interactive Analysis 2 database, we observed that TSP4 expression levels were consistent with overall survival (OS) rate and BC progression, with the highest expression levels observed in the advanced stages of BC and associated with poor OS rate. In our pilot experiments, incubation with recombinant TSP4 promoted the migration and invasion in BC cells. Furthermore, MMP2 expression levels increased after recombinant TSP4 incubation. TSP4-induced-MMP2 expression and cell motility were regulated via the AKT signalling pathway. Our findings facilitate further investigation into TSP4 silencing-based therapeutic strategies for BC.

COMP and TSP-4: Functional Roles in Articular Cartilage and Relevance in Osteoarthritis.

Osteoarthritis (OA) is a slow-progressing joint disease, leading to the degradation and remodeling of the cartilage extracellular matrix (ECM). The usually quiescent chondrocytes become reactivated and accumulate in cell clusters, become hypertrophic, and intensively produce not only degrading enzymes, but also ECM proteins, like the cartilage oligomeric matrix protein (COMP) and thrombospondin-4 (TSP-4). To date, the functional roles of these newly synthesized proteins in articular cartilage are still elusive. Therefore, we analyzed the involvement of both proteins in OA specific processes in in vitro studies, using porcine chondrocytes, isolated from femoral condyles. The effect of COMP and TSP-4 on chondrocyte migration was investigated in transwell assays and their potential to modulate the chondrocyte phenotype, protein synthesis and matrix formation by immunofluorescence staining and immunoblot. Our results demonstrate that COMP could attract chondrocytes and may contribute to a repopulation of damaged cartilage areas, while TSP-4 did not affect this process. In contrast, both proteins similarly promoted the synthesis and matrix formation of collagen II, IX, XII and proteoglycans, but inhibited that of collagen I and X, resulting in a stabilized chondrocyte phenotype. These data suggest that COMP and TSP-4 activate mechanisms to protect and repair the ECM in articular cartilage.

The EGF-LIKE domain of thrombospondin-4 is a key determinant in the development of pain states due to increased excitatory synaptogenesis.

Up-regulation of thrombospondin-4 (TSP4) or voltage-gated calcium channel subunit α2δ1 (Cavα2δ1) proteins in the spinal cord contributes to neuropathic pain development through an unidentified mechanism. We have previously shown that TSP4 interacts with Cavα2δ1 to promote excitatory synaptogenesis and the development of chronic pain states. However, the TSP4 determinants responsible for these changes are not known. Here, we tested the hypothesis that the Cavα2δ1-binding domains of TSP4 are synaptogenic and pronociceptive. We mapped the major Cavα2δ1-binding domains of TSP4 within the coiled-coil and epidermal growth factor (EGF)-like domains in vitro Intrathecal injection of TSP4 fragment proteins containing the EGF-like domain (EGF-LIKE) into naïve rodents was sufficient for inducing behavioral hypersensitivity similar to that produced by an equal molar dose of full-length TSP4. Gabapentin, a drug that binds to Cavα2δ1, blocked EGF-LIKE-induced behavioral hypersensitivity in a dose-dependent manner, supporting the notion that EGF-LIKE interacts with Cavα2δ1 and thereby mediates behavioral hypersensitivity. This notion was further supported by our findings that a peptide within EGF-LIKE (EGFD355-369) could block TSP4- or Cavα2δ1-induced behavioral hypersensitivity after intrathecal injections. Furthermore, only TSP4 proteins that contained EGF-LIKE could promote excitatory synaptogenesis between sensory and spinal cord neurons, which could be blocked by peptide EGFD355-369. Together, these findings indicate that EGF-LIKE is the molecular determinant that mediates aberrant excitatory synaptogenesis and chronic pain development. Blocking interactions between EGF-LIKE and Cavα2δ1 could be an alternative approach in designing target-specific pain medications.

Thrombospondin-4 critically controls transforming growth factor ß1 induced hypertrophic scar formation.

Transforming growth factor ß (TGF-ß) is a growth factor presenting important functions during tissue remodeling and hypertrophic scar (HS) formation. However, the underlying molecular mechanisms are largely unknown. In this study, we identified thrombospondin-4 (TSP-4) as a TGF-ß1 target that essentially mediates TGF-ß1-induced scar formation both in vitro and in vivo. The expression of TSP-4 was compared on both mRNA and protein levels between hypertrophic scar fibroblasts (HSFs) and normal skin fibroblast (NFs) in response to TGF-ß1 treatment. Two signaling molecules, Smad3 and p38, were assessed for their importance in regulating TGF-ß1-mediated TSP-4 expression. The significance of TSP-4 in controlling TGF-ß1-induced proliferation, invasion, migration, and fibrosis in HSFs was analyzed by knocking down endogenous TSP-4 using small hairpin RNA (shRNA) (TSP-4 shRNA). Finally, a skin HS model was established in rats and the scar formation was compared between rats treated with vehicle (saline), TGF-ß1, and TGF-ß1 + TSP-4 shRNA. The TSP-4 level was significantly higher in HSFs than in NFs and TGF-ß1 more potently boosted TSP-4 expression in the former than in the latter. Both Smad3 and p38 essentially mediated TGF-ß1-induced TSP-4 expression. TSP-4 shRNA significantly suppressed TGF-ß1-stimulated proliferation, invasion, migration, or fibrosis of HSFs in vitro and drastically improved wound healing in vivo. TGF-ß1, by activating both Smad3 and p38, induces TSP-4, which in turn not only presents a positive feedback regulation on the activation of Smad3 and p38, but also essentially mediates TGF-ß1-induced HS formation. Targeting TSP-4 thus may benefit HS treatment.

MicroRNA 148a-3p promotes Thrombospondin-4 expression and enhances angiogenesis during tendinopathy development by inhibiting Krüppel-like factor 6.

Tendinopathy is a common musculoskeletal disorder with characteristic hypervascularity. The mechanism of angiogenesis in tendinopathy remains unclear. The present study aimed to investigate the roles of miR-148a-3p in angiogenesis development of tendinopathy. In this study, we demonstrated that miR-148a-3p expression was increased in tendinopathy tissues and positively correlated with CD34 levels which is a specific marker for angiogenesis. We identified Krüppel-like factor 6 (KLF6) as a direct target gene of miR-148a-3p in tenocytes. Furthermore, reduced levels of KLF6 in tendinopathy tissues was showed using qRT-PCR and immunohistochemical analysis, compared with controls. A negative correlation between the levels of KLF6 mRNA and miR-148a-3p was observed. Then, we verified that miR-148a-3p could regulate Tsp-4 expression by targeting KLF6 in tenocyte and was positively correlated with Tsp-4 levels in tendinopathy tissues. In a coculture system of tenocytes with endothelial cells (ECs), we observed that transfection of Lv-miR-148a-3p markedly upregulated EC angiogenesis. In summary, our data establish a novel molecular mechanism by which miR-148a-3p upregulates Tsp-4 expression in tenocytes to promote EC angiogenesis by targeting KLF6, which could be helpful for the treatment of tendinopathy in the future.