Noritaka Matsuo

Dermatopontin interacts with fibronectin, promotes fibronectin fibril formation, and enhances cell adhesion

We report that dermatopontin (DP), an abundant dermal extracellular matrix protein, is found in the fibrin clot and in the wound fluid, which comprise the provisional matrix at the initial stage of wound healing. DP was also found in the serum but at a lower concentration than that in wound fluid. DP co-localized with both fibrin and fibronectin on fibrin fibers and interacted with both proteins. Both normal fibroblast and HT1080 cell adhesion to the fibrin-fibronectin matrix were dose-dependently enhanced by DP, and the adhesion was mediated by α5β1 integrin. The cytoskeleton was more organized in the cells that adhered to the fibrin-fibronectin-DP complex. When incubated with DP, fibronectin formed an insoluble complex of fibronectin fibrils as visualized by electron microscopy. The interacting sites of fibronectin with DP were the first, thirteenth, and fourteenth type III repeats (III1, III13, and III14), with III13 and III14 assumed to be the major sites. The interaction between III2–3 and III12–14 was inhibited by DP, whereas the interaction between I1–5 and III12–14 was specifically and strongly enhanced by DP. Because the interaction between III2–3 and III12–14 is involved in forming a globular conformation of fibronectin, and that between I1–5 and III12–14 is required for forming fibronectin fibrils, DP promotes fibronectin fibril formation probably by changing the fibronectin conformation. These results suggest that DP has an accelerating role in fibroblast cell adhesion to the provisional matrix in the initial stage of wound healing.

Dermatopontin promotes epidermal keratinocyte adhesion via alpha3beta1 integrin and a proteoglycan receptor.

Dermatopontin, an extracellular matrix component initially purified from bovine dermis, promoted cell adhesion of the human epidermal keratinocyte cell line (HaCaT cells). HaCaT cells spread on dermatopontin and formed actin fibers. Adhesion of HaCaT cells to dermatopontin was inhibited by both EDTA and heparin and was mediated in part by alpha3beta1 integrin. A synthetic peptide (DP-4, PHGQVVVAVRS; bovine dermatopontin residues 33-43) specifically inhibited adhesion of cells to dermatopontin, and when the DP-4 peptide was coated on the well, it promoted cell adhesion in a dose-dependent manner. An active core sequence of the DP-4 peptide was localized to an eight-amino acid sequence (GQVVVAVR). These results indicate that dermatopontin is a novel epidermal cell adhesion molecule and suggest that the DP-4 sequence is critical for the cell adhesive activity of dermatopontin. Adhesion of cells to DP-4 was strongly inhibited by heparin. When HaCaT cells were treated with heparitinase I, the cells failed to adhere to DP-4 but chondroitinase ABC treatment did not influence the adhesion activity. DP-4 specifically interacted with biotinylated heparin, and this interaction was inhibited by unlabeled heparin. DP-4 peptide significantly promoted the adhesion of cells overexpressing syndecans, and syndecan bound to a DP-4 peptide affinity column. These results suggest that HaCaT cells adhere to dermatopontin through alpha3beta1 integrin and a heparan sulfate proteoglycan-type receptor, which is likely a syndecan. We conclude that dermatopontin plays a role as a multifunctional adhesion molecule for epidermal cells.

Sp7/Osterix up-regulates the mouse pro-α3(V) collagen gene (Col5a3) during the osteoblast differentiation

Type V collagen is a quantitatively minor collagen, but acts as critical regulator of fibril formation in the extracellular matrix. The purpose of this study is to clarify the mechanism responsible for the transcriptional regulation of the mouse Col5a3 gene in osteoblastic cells. Sp7/Osterix is a transcription factor specifically expressed by osteoblasts and is important for osteoblast differentiation. The overexpression of Sp7/Osterix significantly increased the promoter activity and the endogenous mRNA level of the Col5a3 gene in osteoblastic cells. Conversely, a reduction of Sp7/Osterix by siRNA treatment decreased the promoter activity and the endogenous mRNA level of the Col5a3 gene. A CHIP assay confirmed that Sp7/Osterix interacted with the Col5a3 core promoter in vivo at the Sp1 binding site. The data from the experiments using the osteoblast differentiation model and the co-overexpression of Sp7/Osterix with Sp1 suggest that Sp7/Osterix promotes the expression of the collagen gene, Col5a3, and thereby playing a role in bone formation.

Epiplakin accelerates the lateral organization of keratin filaments during wound healing

BACKGROUNDnEpiplakin (EPPK) belongs to the plakin family of cytolinker proteins and, resembling other members of the plakin family such as BPAG1 (an autoantigen of bullous pemphigoid) and plectin, EPPK has plakin repeat domains (PRDs) that bind to intermediate filaments. Elimination of EPPK by gene targeting in mice resulted in the acceleration of keratinocyte migration during wound healing. EPPK is expressed in proliferating keratinocytes at wound edges and, in view of its putative function in binding to keratin, we postulated that the keratin network in EPPK-null (EPPK(-/-)) mice might be disrupted during wound healing.nnnOBJECTIVEnTo examine this hypothesis and to determine the precise localization of EPPK in relation to keratin filaments, we compared the non-wounded and wounded epidermis of wild-type and EPPK(-/-) mice.nnnMETHODSnNon-wounded epidermis and wounded epidermis from wild-type and EPPK(-/-) mice were examined by immunofluorescence staining and electron microscopy before and after double immunostaining.nnnRESULTSnEPPK was colocalized with keratin 17 (K17) more extensively than with other keratins examined in wounded epidermis. The expression of K5, K10, K6, and K17 was the same in EPPK(-/-) mice after wounding as in normal mice, but diameters of keratin filaments were reduced in EPPK(-/-) keratinocytes. Electron microscopy after immunostaining revealed that EPPK colocalized with K5, K10 and K6 after wounding in wild-type mice.nnnCONCLUSIONnOur data indicate that EPPK accelerates keratin bundling in proliferating keratinocytes during wound healing and suggest that EPPK might contribute to reinforcement of keratin networks under mechanical stress.

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Real time RT-RCR showed that both α1 and α2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-β1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-β receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of α2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.

Dermatopontin Regulates Fibrin Formation and its Biological Activity

Dermatopontin (DP) is a small extracellular matrix component in the dermis. Fibrin is a major component of a provisional matrix that is formed just after wounding. Previously, we found that DP was present in the provisional matrix, and it interacted with fibrin. Here, we examined the role of DP on fibrin function. DP interacted with both the fibrin monomer and fibrils, and was incorporated into the fibrils during fibrin formation. A DP sequence, PHGQVVVAVRS, was identified as a fibrin-binding site, and a globular D domain of fibrin was the binding site for DP. DP accelerated fibrin fibril formation into structurally modified fibrils. Fibrin fibrils formed in the presence of DP enhanced both endothelial cell attachment and cell spreading. The attached cells developed a more organized cytoskeleton when compared with those that attached to fibrin fibrils only. The main receptor for cell adhesion was identified as αvβ3 integrin, and a cooperating receptor was a β1-containing integrin species, probably α5β1 integrin. These results indicate that DP can modify certain biological functions of fibrin, and thus a another function of this extracellular matrix protein was revealed. In addition, the fibrin-DP complex might become useful for developing an improved artificial matrix for improving wound healing.

Long-term administration of the fungus toxin, sterigmatocystin, induces intestinal metaplasia and increases the proliferative activity of PCNA, p53, and MDM2 in the gastric mucosa of aged Mongolian gerbils

ObjectiveThe causal agents of gastric cancer could include fungus toxins. Sterigmatocystin (ST), a fungus toxin, is a risk factor of gastric cancer. We investigated the effects of ST on the stomach tissues of Mongolian gerbils.MethodsSeventy-five-week-old male Mongolian gerbils received ST ad libitum at a concentration of 0xa0ppb (non-treated, nxa0=xa011), 100xa0ppb (nxa0=xa07), or 1,000xa0ppb (nxa0=xa013) dissolved in drinking water for a period of 24xa0weeks. After administration, we tested the histopathological changes and immunostaining for proliferating cell nuclear antigen (PCNA), p53, and MDM2 expression.ResultsWe investigated the histopathological changes and determined the incidence of histopathological changes in animals with various gastric diseases after ST administration at a dose of 0xa0ppb (non-treated control), 100, or 1,000xa0ppb as follows: firstly, indices for gastritis were 18.2, 100, and 100%, those for erosion events were 9.1, 100, and 92.3%, and those for polyps were 0, 71.4, and 61.5%, respectively. These incidences in the ST-administered groups (100 or 1,000xa0ppb) showed significant increases compared with those in the non-treated control group. And, lastly, indices for intestinal metaplasia were 0, 100, and 15.4%, respectively. Furthermore, immunostaining for PCNA, p53, and MDM2 expression showed significantly greater rates in the ST-administered groups (100 or 1,000xa0ppb) than in the non-treated control group.ConclusionThe histopathological and immunohistopathological findings of this study indicate that ST exerts a marked influence on gastric mucus and gland cells, showing dominant gastritis, erosion events, polyps, and intestinal metaplasia in these animals.

Nuclear factor Y (NF-Y) regulates the proximal promoter activity of the mouse collagen α1(XI) gene (Col11a1) in chondrocytes

Type XI collagen, a heterotrimer composed of α1(XI), α2(XI), and α3(XI), plays a critical role in cartilage formation and in skeletal morphogenesis. However, the transcriptional regulation of α1(XI) collagen gene (Col11a1) in chondrocyte is poorly characterized. In this study, we investigated the proximal promoter of mouse Col11a1 gene in chondrocytes. Major transcription start site was located at −299xa0bp upstream of the translation start site, and the proximal promoter lacks a TATA sequence but has a high guanine–cytosine (GC) content. Cell transfection experiments demonstrated that the segment from −116 to −256 is necessary for activation of the proximal Col11a1 promoter, and an electrophoretic mobility shift assay showed that a nuclear protein is bound to the segment from −116 to −176 in this promoter. Additional comparative and in silico analyses demonstrated that an ATTGG sequence, which is critical for binding to nuclear factor Y (NF-Y), is within the highly conserved region from −135 to −145. Interference assays using wild-type and mutant oligonucleotide or with specific antibody revealed that NF-Y protein is bound to this region. Cell transfection experiments with reporter constructs in the absence of NF-Y binding sequence exhibited the suppression of the promoter activity. Furthermore, chromatin immunoprecipitation assay demonstrated that NF-Y protein is directly bound to this region in vivo, and overexpression of dominant-negative NF-Y A mutant also inhibited the proximal promoter activity. Taken together, these results indicate that the transcription factor NF-Y regulates the proximal promoter activity of mouse Col11a1 gene in chondrocytes.

Sp7/Osterix induces the mouse pro-α2(I) collagen gene (Col1a2) expression via the proximal promoter in osteoblastic cells.

Bone is essentially composed of two components, hydroxyapatite and extracellular matrix proteins. The extracellular matrix of bone is primary composed of collagen, mostly type I collagen, with lesser amounts of other types of collagen such as type V collagen. Osteoblast differentiation is a multi-step process in which many classes of factors function in a coordinated manner. Sp7/Osterix, which binds to G/C-rich sequences, is a transcription factor that contributes to osteoblast differentiation. The present study aimed to clarify the involvement of Sp7/Osterix with the proximal promoter region of the mouse Col1a2 gene containing multiple G/C-rich sequences exist. Consequently, a functional analysis of the proximal mouse Col1a2 promoter showed that a substitution mutation of the second G/C-rich sequence from the transcription site specifically decreased the activity of osteoblastic cells. In addition, the experiments of overexpression of Sp7/Osterix and treatment with its specific siRNA showed that this G/C-rich sequence is responsible for the specific expression in osteoblastic cells. Consistent with these data, Sp7/Osterix bound to the region and increased the expression of the Col1a2 gene in association with osteoblast differentiation in the culture system.

Transient expression of mouse pro-α3(V) collagen gene (Col5a3) in wound healing.

The α3(V) chain is poorly characterized among type V collagen chains. Pro-α3(V) collagen is expressed in newly synthesized bone as well as in the superficial fascia of developing muscle. Present study examined the expression in a mouse model of wound healing. Real-time reverse transcriptase polymerase chain reaction and in situ hybridization revealed transient expression of pro-α3(V) chain at a lower level than other fibrillar collagen genes after injury. Immunohistochemistry showed a similar expression pattern in the injured skin. In addition, electron microscopy showed that pro-α3(V) chain was localized in the amorphous nonfibrillar region, but not in fine or dense fibrils. The pro-α3(V) chain co-localized with heparan sulfate, which appeared in the skin after injury and might bind via an acidic segment of the pro-α3(V) chain. The matrix containing the pro-α3(V) chain may therefore be needed for the initiation of wound healing.