Mitsuyasu Kato

Distinct and overlapping patterns of localization of bone morphogenetic protein (BMP) family members and a BMP type II receptor during fracture healing in rats

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are thought to play an important role in bone morphogenesis. The purpose of this study was to determine the locations of BMP-2/-4, osteogenic protein-1 (OP-1, also termed BMP-7), and BMP type II receptor (BMPR-II) during rat fracture healing by immunostaining, and thereby elucidate the possible roles of the BMPs and BMPR-II in intramembranous ossification and endochondral ossification. In the early stage of fracture repair, the expression of BMP-2/-4 and OP-1 was strongly induced in the thickened periosteum near the fracture ends, and coincided with an enhanced expression of BMPR-II. On day 7 after fracture, staining for BMP-2/-4 and OP-1 immunostaining was increased in various types of chondrocytes, and was strong in fibroblast-like spindle cells and proliferating chondrocytes in endochondral bone. On day 14 after fracture, staining with OP-1 antibody disappeared in proliferating and mature chondrocytes, while BMP-2/-4 staining continued in various types of chondrocytes until the late stage. In the newly formed trabecular bone, BMP-2/-4 and OP-1 were present at various levels. BMPR-II was actively expressed in both intramembranous ossification and endochondral ossification. Additionally, immunostaining for BMP-2/-4 and OP-1 was observed in multinucleated osteoclast-like cells on the newly formed trabecular bone, along with BMPR-II. In reference to our previous study of BMP type I receptors (BMPR-IA and BMPR-IB), BMPR-II was found to be co-localized with BMPR-IA and BMPR-IB. BMP-2/-4 and OP-1 antibodies exhibited distinct and overlapping immunostaining patterns during fracture repair. OP-1 may act predominantly in the initial phase of endochondral ossification, while BMP-2/-4 acts throughout this process. Thus, these findings suggested that BMPs acting through their BMP receptors may play major roles in modulating the sequential events leading to bone formation.

Focal Adhesion Kinase Activity Is Required for Bone Morphogenetic Protein—Smad1 Signaling and Osteoblastic Differentiation in Murine MC3T3-E1 Cells

Cell‐matrix interactions via integrins are essential for osteoblastic differentiation. We have shown that signals activated by aggregation of α2β1‐integrin with type I collagen are involved in the differentiation of osteoblastic MC3T3‐E1 cells. Focal adhesion kinase (FAK) is an immediate downstream signal of the β1‐integrin, and inactivation of FAK has been shown to disrupt osteoblastic differentiation. To elucidate roles of FAK in osteoblastic cells, we examined MC3T3‐E1 cells stably expressing antisense FAK (asFAK) messenger RNA (mRNA). Alkaline phosphatase (ALP) activity, an osteoblastic marker, did not increase in asFAK cells with a long‐term culture until 21 days or in response to bone morphogenetic protein 2 (BMP‐2). Treatment with BMP‐2 also failed to stimulate the expression of osteocalcin in asFAK cells. In control MC3T3‐E1 cells, BMP‐2 induced translocation of Smad1 into nuclei to stimulate transcriptional activity of the Smad6 promoter gene that contains a Smad1 response element. In contrast, BMP‐2 failed to increase transcriptional activity of Smad6 promoter gene in asFAK cells, although it induced nuclear translocation of Smad1. These results indicate that FAK was involved in Smad1‐dependent transcriptional activity but not in nuclear translocation of Smad1 in osteoblastic cells. Hence, FAK activation by integrins might converge transcriptional activation by BMP of its target genes in osteoblastic cells. These observations suggest that FAK activity is essential for BMP‐Smad signaling to stimulate osteoblastic differentiation.

Expression and localization of bone morphogenetic proteins (BMPs) and BMP receptors in ossification of the ligamentum flavum

To clarify the pathogenesis of ossification of the ligamentum flavum (OLF), we examined the expression and localization of bone morphogenetic proteins (BMPs) and their receptors (BMPRs) in the ligamentum flavum of the patients with OLF by immunohistochemical staining and compared them with staining patterns in control patients. The BMPRs appeared extensively in mature and immature chondrocytes around the calcified zone and in spindle-shaped cells and round cells in the remote part from ossified foci in examined tissue of OLF. The ligands for BMPRs, BMP-2/-4 and osteogenic protein-1 (OP-1)/BMP-7, colocalized in OLF patients. In the control cases, expression of BMPs and BMPRs was observed around the calcified zone at the insertion of the ligamentum flavum to the bone, and limited expression was found in the smaller range. Thus, the expression profile of BMPs and BMPRs in OLF patients was entirely different from the control patients, suggesting that BMPs may be involved in promoting endochondral ossification at ectopic ossification sites in OLF, and that ossification activity is continuous in these patients.

Antiangiogenic Agent (TNP-470) Inhibition of Ectopic Bone Formation Induced by Bone Morphogenetic Protein-2

Bone morphogenetic protein (BMP) is a potent inducer of ectopic bone formation, and TNP-470, a synthetic analog of fumagillin, is an antiangiogenic agent that strongly inhibits neovascular formation in vivo. We investigated the effects of TNP-470 on BMP-induced ectopic bone formation to clarify the role of angiogenesis in bone formation. Collagen pellets containing recombinant human BMP-2 (rhBMP-2) were implanted beneath the fasciae of dorsal muscles in mice. By daily subcutaneous administration of TNP-470, ectopic new bone formation was inhibited in a dose-dependent manner. Histological examination revealed that TNP-470 prevented proliferation of mesenchymal cells and chondrogenesis at the initial step of endochondral bone formation. Immunohistochemical staining with a specific antibody against bone morphogenetic protein type IA receptor showed that TNP-470 reduced the number of receptor-positive cells surrounding the BMP pellets. The inhibitory effect of TNP-470 on bone formation continued during the period of its administration, and discontinuation of treatment was followed by the resumption of the whole process of endochondral bone formation. This study showed that TNP-470 reversibly inhibits the biological activity of rhBMP-2 in the early stage of bone induction, suggesting that angiogenesis may play an essential role in the recruitment of BMP-receptor-positive cells that can respond to rhBMP-2 and differentiate into chondrocytes and/or osteoblasts.

Cell Density-dependent Apoptosis in HL-60 Cells, Which Is Mediated by an Unknown Soluble Factor, Is Inhibited By Transforming Growth Factor β1 and Overexpression of Bcl-2

We report a novel mode of apoptosis induction observed in human leukemic HL-60 cells. These cells spontaneously underwent apoptosis in the course of proliferation when the cell density became higher than 1 × 106/ml. This occurred under ordinary in vitro culture conditions, with or without fetal calf serum. Even the low density cells were committed to undergo apoptosis if they were cultured under artificially concentrated conditions. Replacement of the culture supernatant of the low density cells by that of the high density ones resulted in apoptosis induction in the former cells. This apoptosis-inducing activity of the high density cell culture supernatant was completely eliminated by the action of trypsin but was fully restored following ultrafiltration by 3-kDa pore-sized membrane. A strong apoptosis-inducing activity was recovered from the culture supernatant of the high density HL-60 cells at a specific fraction in reverse-phase column chromatography. Neither an interleukin-β converting enzyme inhibitor nor CPP-32 inhibitor blocked the induction of cell density-dependent apoptosis in HL-60 cells, although overexpression of Bcl-2 protein markedly attenuated the induction of this mode. Surprisingly, transforming growth factor-β1 and activin A did not induce but, rather, inhibited the induction of cell density-dependent apoptosis. These data suggest that HL-60 cells release an unknown low molecular weight peptide-containing factor in response to an increase in cell density to induce apoptosis in an autocrine manner and that the interleukin-β converting enzyme-independent intracellular machinery for this mode of apoptosis is strongly affected by signaling events through the transforming growth factor-β1 receptor and by the action of Bcl-2 oncoprotein.

Bone morphogenetic protein type IB receptor is progressively expressed in malignant glioma tumours.

The distribution of bone morphogenetic protein (BMP) type I receptors and the activin type I receptor (ActR-I) was investigated in 16 cases of human glioma and five cases of non-tumourous gliosis tissue by immunohistochemical technique. Both BMP type IA (BMPR-IA) and the type IB (BMPR-IB) receptors were detected in human glioma cells. A significant increase in BMPR-IB in tumour cells was observed in malignant glioma compared with both low-grade astrocytomas (n=16, P<0.005) and gliosis (n=13, P<0.001). However, enhancement of BMPR-IA staining was moderate and ActR-I staining was only weakly expressed in the malignant glioma tumours. Osteogenic protein (OP)-1/BMP-7, which is known to bind BMPR-IA, BMPR-IB and ActR-I, was expressed in nervous tissue and was also detected in anaplastic areas of malignant glioma. In contrast to the tissue materials, BMPR-IA was expressed to a stronger degree than BMPR-IB in human glioma cell lines; the growth of these cells was suppressed by OP-1. These results suggest the presence of BMP receptors and a functional role for BMPs in malignant glioma.

Expression of tetra‐spans transmembrane family (CD9, CD37, CD53, CD63, CD81 and CD82) in normal and neoplastic human keratinocytes: an association of CD9 with α3β1 integrin

Tetra‐spans transmembrane family (TSTF) members (CD9, CD37, CD53, CD63, CD81 and CD82) have potent effects on cell growth, motility and adhesion in various cells. However, little is known about their expression in human skin. Using immunohistological techniques, we have studied the localization of all six members of TSTF in normal and carcinomatous human keratinocytes. CD9, CD81 and CD82 were expressed in the entire living layers of the epidermis. Their staining pattern was quite similar, and was mainly intercellular with occasional intracellular immunoreactivity. CD53 expression was confined to the intercellular spaces of the upper spinous or granular layer in the normal epidermis. No clear‐cut expression of CD63 could be detected in the epidermis. CD37 was not detected at all. Cultured human keratinocytes also expressed CD9, CD81 and CD82 at the surface membrane of cell‐cell boundaries. Expression of CD37 and CD53 was negative in cultured keratinocyte, while CD63 was clearly localized in the cytoplasmic lysosomes. An immunoprecipitation assay revealed that α3β1 integrin is molecularly associated with CD9. The expression of CD9, CD81 and CD82 was markedly down‐regulated in basal cell carcinoma but not in Bowens disease. The abundant and differential expression of TSTF molecules and the selective association of CD9 with α3β1 integrin suggest that the TSTF molecules may be involved in the regulation of epidermal differentiation and integrity in vivo.

EXPRESSION OF TGF-BETA TYPE I AND TYPE II RECEPTORS IN RAT EYES

Transforming growth factor beta (TGF-beta) transduces signals through mediation of type I and type II serine/threonine kinase receptors. The expression of TGF-beta type I (T beta R-I) and II (T beta R-II) receptors in rat eyes was investigated immunohistochemically. T beta R-I and T beta R-II immunoreactivity was detected in corneal and conjunctival epithelial cells, corneal endothelial cells, ciliary epithelial cells, lens epithelial cells, retinal pigment epithelial cells, and choroidal vessels. This co-expression of T beta R-I and T beta R-II indicates that the above cells respond to TGF-beta and, because TGF-beta is reported to be produced in ocular tissues, that it may have important autocrine and/or paracrine roles in the growth and metabolism of ocular tissues in situ.

Distribution of Transforming Growth Factor‐β and Its Receptors in Gastric Carcinoma Tissue

The distribution of the three mammalian isoforms of transforming growth factor (TGF)‐β (TGF‐β1,‐β2, and ‐β3) as well as their signaling receptors, TGF‐β type I and type II receptors (TβR‐I and TβR‐II, respectively), in gastric carcinoma tissue was examined by immunohistochemistry using specific antibodies. Tissue specimens were obtained from 25 cases of gastric carcinoma, which were classified into two groups according to Laurens classification, i.e. 15 cases of diffuse carcinoma and 10 cases of intestinal carcinoma. In normal gastric mucosa apart from carcinoma nests, all of TGF‐β1, ‐β2, ‐β3, TβR‐I and TβR‐II were clearly demonstrated in fundic glands. In sharp contrast, none of them was detectable in surface mucous cells. In carcinoma cells, strong staining for TGF‐β1, ‐β2 and β3 was obtained only in diffuse‐type carcinoma. In particular, carcinoma cells scattered as single cells or small nests had a tendency to show strong staining for TGF‐βs. The receptors tended to be distributed concomitantly with the ligands, and diffuse‐type carcinoma showed stronger receptor staining than intestinal‐type carcinoma. In cancer stroma, TGF‐βs and receptors were detected in both diffuse and intestinal types, but the area with positive staining was wider and more dispersed in diffuse‐type carcinoma than in intestinal carcinoma. These results suggest that TGF‐β may contribute in part to the variety of histogenesis and mode of progression of gastric carcinoma.

Differential localization of TGF-β-precursor isotypes in normal human skin

Abstract Transforming growth factor-β (TGF-β) can act as a multi-functional regulator of both cell growth and differentiation. Three isotypes of TGF-βs namely TGF-β1, TGF-β2 and TGF-β3, have been found in human tissues. Up to now, little is known about the distribution patterns of the TGF-β isotypes in human skin. Using the TGF-β-precursor (latency-associated peptides) specific antibodies to confirm the specificity, we studied the immunohistochemical distribution of TGF-β1–3 in human skin. TGF-β2 was found mainly in the intercellular space of all the layers of the epidermis as well as in the cytoplasm with a weak staining. In contrast, TGF-β3 was present in the subepidermal area of the dermis. TGF-β1 was observed obviously in neither epidermis nor dermis. These results showed the differential localization of TGF-β isotypes in human skin, suggesting that the TGF-β2 and TGF-β3 may regulate the human skin function in an epithelial autocrine or mesenchymal-epithelial interaction manner.