The expression of two collagen receptor subfamilies, integrins and discoidin domains during osteogenic and chondrogenic differentiation of human mesenehymal stem cells.

BACKGROUND: Collagen receptors are characterized by binding to and being activated by collagens. We know little about the molecular mechanism by which the integrins and discoidin domains (DDRs) recognize collagen. OBJECTIVE: The aim of this study was to investigate the expression of two main collagen receptor subfamilies, integrins and DDRs, during osteogenic and chondrogenic differentiation of human mesenehymal stem cells (hMSCs). METHODS: Using qRT-PCR, Western blots and FACS, the levels of DDR1, DDR2, integrin subunits ß1, α1, α2, α10 and α11 receptors on hMSCs, were assessed upon activation by collagen type I, as well as during osteogenic and chondrogenic differentiation. RESULTS: The expression of DDR2 and integrin α11ß1 was altered compared with other receptors when the cells were cultured under undifferentiated conditions. During osteogenic and chondrogenetic differentiation, DDR2 and α11 were up-regulated during early stages (6 day) of osteogenesis and chondrogenesis, respectively. The expression and activation of DDR2 was concomitant with another receptor integrin subunit ß1 during osteogenetic differentiation. CONCLUSIONS: The results suggested that DDR2 was more specific for osteogenesis than chondrogenesis, while integrin α11ß1 was more specific in chondrogenesis. DDR2 and α11 may play a role in the regulation of osteogenesis and chondrogenesis based on the differential expression of these receptors during lineage-dependent changes.

An epidermal growth factor motif of developmental endothelial locus 1 protein inhibits efficient angiogenesis in explanted squamous cell carcinoma in vivo

ABSTRACT Background: Cancer gene therapy using a nonviral vector is expected to be repeatable, safe, and inexpensive, and to have long-term effectiveness. Gene therapy using the E3 and C1 (E3C1) domain of developmental endothelial locus-1 (Del1) has been shown to improve prognosis in a mouse transplanted tumor model. Objective: In this study, we examined how this treatment affects angiogenesis in mouse transplanted tumors. Materials and methods: Mouse transplanted tumors (SCCKN human squamous carcinoma cell line) were injected locally with a nonviral plasmid vector encoding E3C1 weekly. Histochemical analysis of the transplanted tumors was then performed to assess the effects of E3C1 on prognosis. Results: All mice in the control group had died or reached an endpoint within 39 days. In contrast, one of ten mice in the E3C1 group had died by day 39, and eight of ten had died or reached an endpoint by day 120 (p < 0.01). Enhanced apoptosis in tumor stroma was seen on histochemical analyses, as was inhibited tumor angiogenesis in E3C1-treated mice. In addition, western blot analysis showed decreases in active Notch and HEY1 proteins. Conclusion: These findings indicate that cancer gene therapy using a nonviral vector encoding E3C1 significantly improved life-span by inhibiting tumor angiogenesis. (REV INVEST CLIN. 2021;73(1):39-51)

An Epidermal Growth Factor Motif of Developmental Endothelial Locus 1 Protein Inhibits Efficient Angiogenesis in Explanted Squamous Cell Carcinoma In Vivo.

BACKGROUND: Cancer gene therapy using a nonviral vector is expected to be repeatable, safe, and inexpensive, and to have longterm effectiveness. Gene therapy using the E3 and C1 (E3C1) domain of developmental endothelial locus-1 (Del1) has been shown to improve prognosis in a mouse transplanted tumor model. OBJECTIVE: In this study, we examined how this treatment affects angiogenesis in mouse transplanted tumors. MATERIALS AND METHODS: Mouse transplanted tumors (SCCKN human squamous carcinoma cell line) were injected locally with a nonviral plasmid vector encoding E3C1 weekly. Histochemical analysis of the transplanted tumors was then performed to assess the effects of E3C1 on prognosis. RESULTS: All mice in the control group had died or reached an endpoint within 39 days. In contrast, one of ten mice in the E3C1 group had died by day 39, and eight of ten had died or reached an endpoint by day 120 (p < 0.01). Enhanced apoptosis in tumor stroma was seen on histochemical analyses, as was inhibited tumor angiogenesis in E3C1-treated mice. In addition, western blot analysis showed decreases in active Notch and HEY1 proteins. CONCLUSION: These findings indicate that cancer gene therapy using a nonviral vector encoding E3C1 significantly improved life-span by inhibiting tumor angiogenesis.

Discoidin domain receptors: Micro insights into macro assemblies.

Assembly of cell-surface receptors into specific oligomeric states and/or clusters before and after ligand binding is an important feature governing their biological function. Receptor oligomerization can be mediated by specific domains of the receptor, ligand binding, configurational changes or other interacting molecules. In this review we summarize our understanding of the oligomeric state of discoidin domain receptors (DDR1 and DDR2), which belong to the receptor tyrosine kinase family (RTK). DDRs form an interesting system from an oligomerization perspective as their ligand collagen(s) can also undergo supramolecular assembly to form fibrils. Even though DDR1 and DDR2 differ in the domains responsible to form ligand-free dimers they share similarities in binding to soluble, monomeric collagen. However, only DDR1b forms globular clusters in response to monomeric collagen and not DDR2. Interestingly, both DDR1 and DDR2 are assembled into linear clusters by the collagen fibril. Formation of these clusters is important for receptor phosphorylation and is mediated in part by other membrane components. We summarize how the oligomeric status of DDRs shares similarities with other members of the RTK family and with collagen receptors. Unraveling the multiple macro-molecular configurations adopted by this receptor-ligand pair can provide novel insights into the intricacies of cell-matrix interactions.

Structural mapping of hot spots within human CASPR2 discoidin domain for autoantibody recognition.

Accumulating evidence has showed that anti-CASPR2 autoantibodies occur in a long list of neurological immune disorders including limbic encephalitis (LE). Belonging to the well-known neurexin superfamily, CASPR2 has been suggested to be a central node in the molecular networks controlling neurodevelopment. Distinct from other subfamilies in the neurexin superfamily, the CASPR subfamily features a unique discoidin (Disc) domain. As revealed by our and others' recent studies, CASPR2 Disc domain bears a major epitope for autoantibodies. However, structural information on CASPR2 recognition by autoantibodies has been lacking. Here, we report the crystal structure of human CASPR2 Disc domain at a high resolution of 1.31 Å, which is the first atomic-resolution structure of the CASPR subfamily members. The Disc domain adopts a total ß structure and folds into a distorted jellyroll-like barrel with a conserved disulfide-bond interlocking its N- and C-termini. Defined by four loops and located in one end of the barrel, the "loop-tip surface" is totally polar and easily available for protein docking. Based on structure-guided epitope prediction, we generated nine mutants and evaluated their binding to autoantibodies of cerebrospinal fluid from twelve patients with limbic encephalitis. The quadruple mutant G69N/A71S/S77N/D78R impaired CASPR2 binding to autoantibodies from eleven LE patients, which indicates that the loop L1 in the Disc domain bears hot spots for autoantibody interaction. Structural mapping of autoepitopes within human CASPR2 Disc domain sheds light on how autoantibodies could sequester CASPR2 ectodomain and antagonize its functionalities in the pathogenic processes.

The C. elegans Discoidin Domain Receptor DDR-2 Modulates the Met-like RTK-JNK Signaling Pathway in Axon Regeneration.

The ability of specific neurons to regenerate their axons after injury is governed by cell-intrinsic regeneration pathways. However, the signaling pathways that orchestrate axon regeneration are not well understood. In Caenorhabditis elegans, initiation of axon regeneration is positively regulated by SVH-2 Met-like growth factor receptor tyrosine kinase (RTK) signaling through the JNK MAPK pathway. Here we show that SVH-4/DDR-2, an RTK containing a discoidin domain that is activated by collagen, and EMB-9 collagen type IV regulate the regeneration of neurons following axon injury. The scaffold protein SHC-1 interacts with both DDR-2 and SVH-2. Furthermore, we demonstrate that overexpression of svh-2 and shc-1 suppresses the delay in axon regeneration observed in ddr-2 mutants, suggesting that DDR-2 functions upstream of SVH-2 and SHC-1. These results suggest that DDR-2 modulates the SVH-2-JNK pathway via SHC-1. We thus identify two different RTK signaling networks that play coordinated roles in the regulation of axonal regeneration.

[Expression and significance of discoidin domain receptor 1 in salivary gland mucoepidermoid carcinoma].

PURPOSE: To investigate the expression and significance of discoidin domain receptor 1 (DDR1) in salivary gland mucoepidermoid carcinoma (MEC). METHODS: Immunohistochemical and Western blot method were used to detect the expression of DDR1 in MEC M3SP2 and MC3 cell lines. Immunohistochemical method was used to detect the expression of DDR1 in 58 MEC and 20 normal salivary gland tissues. SPSS 13.0 software package was used for statistical analysis. RESULTS: The positive expression rate of DDR1 in salivary gland MEC tissues was 79.3%, significantly higher than 10.0% in normal salivary gland tissues (P<0.01). High expression of DDR1 was not related to clinicopathological parameters of MEC(P>0.05). The expression of DDR1 was positive in MEC M3SP2 and MC3 cell lines. CONCLUSIONS: DDR1 may play an important role in carcinogenesis and progress of salivary gland MEC.

Discoidin Domains as Emerging Therapeutic Targets.

Discoidin (DS) domains are found in eukaryotic and prokaryotic extracellular and transmembrane multidomain proteins. These small domains play different functional roles and can interact with phospholipids, glycans, and proteins, including collagens. DS domain-containing proteins are often involved in cellular adhesion, migration, proliferation, and matrix-remodeling events, while some play a major role in blood coagulation. Mutations in DS domains have been associated with various disease conditions. This review provides an update on the structure, function, and modulation of the DS domains, with a special emphasis on two circulating blood coagulation cofactors, factor V and factor VIII, and the transmembrane neuropilin receptors that have been targeted for inhibition by biologics and small chemical compounds.

[Repair effects of close-to-bone needing combined with electroacupuncture on extracellular matrix of cartilage in rabbits with knee osteoarthritis].

OBJECTIVE: To observe the effects of close-to-bone needing combined with electroacupuncture (EA) on cartilage collagen type Ⅱ/discoidin domain receptor/matrix metalloproteinase 13 (collagen type Ⅱ/DDR2/MMP 13) signaling pathway in rabbits with knee osteoarthritis (KOA), and to explore the possible action mechanism of this method on repair of extracellular matrix of knee cartilage. METHODS: Forty New Zealand white rabbits were randomly assigned into a normal group (10 rabbits) and a model establishing group (30 rabbits). The Hulth-Telhag technique was applied to establish the model of KOA, and X-ray was used for outcome assessment. The rabbits with successful modeling were randomly assigned into a model group, a close-to-bone needing group, a regular acupuncture group, 10 rabbits in each one. The rabbits in the close-to-bone needing group were treated with close-to-bone needing and EA; the rabbits in the regular acupuncture group were treated with regular acupuncture and EA. "Neixiyan" (EX-LE 4), "Dubi" (ST 35), "Yinlingquan" (SP 9), "Zusanli" (ST 36) and "Liangqiu" (ST 34) were selected in the two groups. The intervention was given for 20 min, once a day; the intervention of 5 days made 1 session, 2 days as the interval and totally 4 sessions were given. Rabbits in normal and model group were immobilized without any treatment. After the treatment, western blotting method was applied to evaluate the expression of DDR2 and collagen type Ⅱ; the activity of collagen type Ⅱ, DDR2 and MMP 13 was assessed by immunohistochemistry method; the mRNA expression of DDR2 and MMP 13 was determined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Compared with the normal group, the activity expression of collagen type Ⅱ were significantly reduced in the other groups (all P<0.01),while the activity and mRNA expression of DDR2 and MMP 13 were notably increased (all P<0.01). Compared with the model group, the activity expression of collagen type Ⅱ in the close-to-bone needing group and regular acupuncture group were increased (all P<0.01), while the activity and mRNA expression of DDR2 and MMP 13 were reduced (all P<0.01). Compared with the regular acupuncture group, the activity and mRNA expression of MMP 13 and DDR2 in the close-to-bone needing group were reduced (all P<0.01), while the activity expression of collagen type Ⅱ were increased (P<0.01). CONCLUSIONS: The close-to-bone needing combined with EA and regular EA could both promote the repair of knee cartilage, where closing-to-bone needing combined with EA shows a superior efficacy. The mechanism may be associated with the blocking effect of collagen type Ⅱ/DDR2/MMP13 signaling pathway and the inhibiting effect of degradation in extracellular matrix of cartilage.