Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction.

Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF-dependent bone marrow macrophages (M-BMM phi) appeared within 3 d. Tartrate-resistant acid phosphatase-positive osteoclasts were also formed when M-BMM phi were further cultured for 3 d with mouse tumor necrosis factor alpha (TNF-alpha) in the presence of M-CSF. Osteoclast formation induced by TNF-alpha was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti-RANK (ODF/RANKL receptor) antibody. Experiments using M-BMM phi prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-alpha. Osteoclasts induced by TNF-alpha formed resorption pits on dentine slices only in the presence of IL-1alpha. These results demonstrate that TNF-alpha stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL-RANK system. TNF-alpha together with IL-1alpha may play an important role in bone resorption of inflammatory bone diseases.

A human alpha-fetoprotein enhancer-binding protein, ATBF1, contains four homeodomains and seventeen zinc fingers.

We have isolated a full-length cDNA encoding a protein (ATBF1) that binds to an AT-rich motif in the human alpha-fetoprotein gene enhancer. The amino acid sequence deduced from the cDNA revealed that this is the largest DNA-binding protein (306 kDa) known to date, containing four homeodomains, 17 zinc finger motifs, and a number of segments potentially involved in transcriptional regulation. Although the exact function of this protein has not been determined, these structural features suggest that ATBF1 plays a transcriptional regulatory role.

Possible roles of arachidonic acid and its metabolites in induction of tissue plasminogen activator (t-PA) production in human fibroblast, IMR-90 cells by proteose peptone.

Proteose peptone (p.peptone) had an ability to induce tissue plasminogen activator (t-PA) production by human embryonic lung fibroblast, IMR-90 cells. We previously demonstrated that the induction was closely related to the activation of phospholipase A2 in the cells stimulated by p.peptone. In this report, we describe the involvement of arachidonate metabolism in the induction. The induction was inhibited in a dose-dependent manner by 5,8,11,14-eicosatetraenoic acid (ETYA), an inhibitor of both cycloxygenase and lypoxygenase, and also by nordihydroguaiaretic acid (NDGA), which in low concentrations selectively inhibits lipoxygenase. However, indomethacin, a specific inhibitor of cycloxygenase, had no effect on the induction. 5-hydroxyeicosatetraenoic acid (5-HETE), which is an arachidonate metabolite derived from lipoxygenase pathway, had an inductive effect, but prostaglandin E1 (PGE1), which is a metabolite from cycloxygenase pathway, had no effect on t-PA production by the cells. These results suggest that arachidonate metabolism is involved in the induction of t-PA production in IMR-90 cells by p.peptone, and that arachidonate metabolite(s) from lipoxygenase pathway is responsible for the induction.

Identification of heparin-binding stretches of a naturally occurring deleted variant of hepatocyte growth factor (dHGF).

A deleted variant of hepatocyte growth factor (dHGF) is a naturally occurring major variant of HGF, which lacks five consecutive amino acid residues in the first kringle domain. While both HGF and dHGF bind to heparin, the residues involved in the binding to heparin have not been identified in either protein. To identify the residues involved in the binding, we made a series of dHGF mutants in which basic residues in the N-terminal and the first kringle domains were replaced with alanine residue. The analysis of heparin-binding ability revealed that three stretches, 42RCTRNK in the hairpin loop structure, and 2RKRR and 27KIKTKK in the N-terminal basic region, are involved in the binding. Alanine substitution of each basic residue except 3K and 27K in the stretches reduced the heparin-binding ability of dHGF, and the decrease was additive. Conversely, lysine substitution of 37D, 38Q or 64Q in the N-terminal domain increased heparin-binding ability. These results suggest that stretches distant from each other in the primary structure come into close proximity when the polypeptide folds into protein, and form a heparin-binding site with clusters of basic residues.

Role of Ca2+ in t-PA production by human embryonic lung diploid fibroblast, IMR-90 cells, stimulated by proteose peptone.

Proteose peptone (p.peptone) remarkably induced tissue plasminogen activator (t-PA) activity in the conditioned medium of confluently cultured human embryonic lung diploid fibroblast, IMR-90 cells, in a dose-dependent manner. t-PA activity correlated well with the amount of t-PA antigen found in the conditioned medium of IMR-90 cells stimulated by p.peptone. t-PA production by IMR-90 cells stimulated by p.peptone was dependent on extracellular Ca2+ concentration and maximum t-PA production required approximately 3.6 mM extracellular Ca2+. Conversely, elimination of Ca2+ from the culture medium by EGTA, Ca2+ chelate agent, strongly inhibited t-PA production induced by p.peptone. t-PA production induced by p.peptone was inhibited in a dose-dependent manner by Verapamil, which inhibits Ca2+ uptake through the slow channels and also by W-7, an inhibitor of calmodulin. These results suggested that influx of extracellular Ca2+ into IMR-90 cells was caused by p.peptone and induced t-PA production by the cells.

Immunological characterization of circulating osteoprotegerin/osteoclastogenesis inhibitory factor: increased serum concentrations in postmenopausal women with osteoporosis.

Osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) is a soluble member of the tumor necrosis factor receptor family of proteins and plays an important role in the negative regulation of osteoclastic bone resorption. Whether OPG/OCIF circulates in human blood and how its level changes under pathological conditions is not known. To address these issues, a panel of monoclonal antibodies was generated against recombinant OPG/OCIF and screened for reactivity with solid-phase monomeric and homodimeric forms of the recombinant protein. Antibodies that showed high affinity for both forms of OPG/OCIF and those that selectively recognized the homodimer were identified, enabling development of two types of sensitive enzyme-linked immunosorbent assay (ELISA): one that detects both forms of OPG/OCIF equally and one specific for the homodimer. Characterization of circulating OPG/OCIF with these ELISAs revealed that the protein exists in human serum mainly in the monomeric form. The serum concentration of OPG/OCIF increased with age in both healthy Japanese men and women, and was significantly higher in postmenopausal women with osteoporosis than in age-matched controls. Within the osteoporotic group, serum OPG/OCIF concentrations were higher in patients with low bone mass. Serum OPG/OCIF concentrations were also significantly increased in those postmenopausal women with a high rate of bone turnover, as determined by increased serum bone-specific alkaline phosphatase and urinary excretion of pyridinoline and deoxypyridinoline. The results suggested that circulating OPG/OCIF levels are regulated by an age-related factor(s) and that the increased serum concentration may reflect a compensative response to enhanced osteoclastic bone resorption and the resultant bone loss rather than a cause of osteoporosis.

Transforming growth factor beta affects osteoclast differentiation via direct and indirect actions.

Transforming growth factor beta (TGF-beta) is abundant in bone and has complex effects on osteolysis, with both positive and negative effects on osteoclast differentiation, suggesting that it acts via more than one mechanism. Osteoclastogenesis is determined primarily by osteoblast (OB) expression of the tumor necrosis factor (TNF)-related molecule receptor activator of NF-kappaB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), which are increased and decreased, respectively, by osteolytic factors. A RANKL-independent osteoclastogenic mechanism mediated by TNF-alpha has also been shown. Therefore, we investigated TGF-beta effects on osteoclast formation in culture systems in which osteoclastogenic stimulus is dependent on OBs and culture systems where it was provided by exogenously added RANKL or TNF-alpha. Both OPG and TGF-beta inhibited osteoclast formation in hemopoietic cell/OB cocultures, but the kinetics of their action differed. TGF-beta also inhibited osteoclastogenesis in cocultures of cells derived from OPG null (opg-/-) mice. TGF-beta strongly decreased RANKL messenger RNA (mRNA) expression in cultured osteoblasts, and addition of exogenous RANKL to TGFbeta-inhibited cocultures of opg-/- cells partially restored osteoclastogenesis. Combined, these data indicate that the inhibitory actions of TGF-beta were mediated mainly by decreased OB production of RANKL. In contrast, in the absence of OBs, TGF-beta greatly increased osteoclast formation in recombinant RANKL- or TNF-alpha-stimulated cultures of hemopoietic cells or RAW 264.7 macrophage-like cells to levels several-fold greater than attainable by maximal stimulation by RANKL or TNF-alpha. These data suggest that TGF-beta may increase osteoclast formation via action on osteoclast precursors. Therefore, although RANKL (or TNF-alpha) is essential for osteoclast formation, factors such as TGF-beta may powerfully modify these osteoclastogenic stimuli. Such actions may be critical to the control of physiological and pathophysiological osteolysis.

Osteoprotegerin produced by osteoblasts is an important regulator in osteoclast development and function.

Osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoclast differentiation factor, inhibits both differentiation and function of osteoclasts. We previously reported that OPG-deficient mice exhibited severe osteoporosis caused by enhanced osteoclastic bone resorption. In the present study, potential roles of OPG in osteoclast differentiation were examined using a mouse coculture system of calvarial osteoblasts and bone marrow cells prepared from OPG-deficient mice. In the absence of bone-resorbing factors, no osteoclasts were formed in cocultures of wild-type (+/+) or heterozygous (+/-) mouse-derived osteoblasts with bone marrow cells prepared from homozygous (-/-) mice. In contrast, homozygous (-/-) mouse-derived osteoblasts strongly supported osteoclast formation in the cocultures with homozygous (-/-) bone marrow cells, even in the absence of bone-resorbing factors. Addition of OPG to the cocultures with osteoblasts and bone marrow cells derived from homozygous (-/-) mice completely inhibited spontaneously occurring osteoclast formation. Adding 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] to these cocultures significantly enhanced osteoclast differentiation. In addition, bone-resorbing activity in organ cultures of fetal long bones derived from homozygous (-/-) mice was markedly increased, irrespective of the presence and absence of bone-resorbing factors, in comparison with that from wild-type (+/+) mice. Osteoblasts prepared from homozygous (-/-), heterozygous (+/-), and wild-type (+/+) mice constitutively expressed similar levels of RANKL messenger RNA, which were equally increased by the treatment with 1alpha,25(OH)2D3. When homozygous (-/-) mouse-derived osteoblasts and hemopoietic cells were cocultured, but direct contact between them was prevented, no osteoclasts were formed, even in the presence of 1alpha,25(OH)2D3 and macrophage colony-stimulating factor. These findings suggest that OPG produced by osteoblasts/stromal cells is a physiologically important regulator in osteoclast differentiation and function and that RANKL expressed by osteoblasts functions as a membrane-associated form.

Hypocalcemic effect of osteoclastogenesis inhibitory factor/osteoprotegerin in the thyroparathyroidectomized rat.

Osteoclastogenesis inhibitory factor (OCIF), also termed as osteoprotegerin (OPG), is a soluble member of the tumor necrosis factor receptor family. Although OCIF/OPG is shown to inhibit osteoclast formation in vitro and prevent ovariectomy-induced bone loss in vivo, its effect on serum calcium level remains to be determined. In this study we examined the acute effect of OCIF on thyroparathyroidectomized rats whose serum calcium concentrations were raised either by exogenous PTH or 1,25-(OH)2D3. When OCIF was administered at the start of PTH infusion, it attenuated the initial rise in serum calcium. When OCIF was administered into rats with established hypercalcemia, it decreased serum calcium rapidly (within 2 hr) and dramatically. OCIF did not increase urinary calcium excretion. These findings, especially the rapid onset of its hypocalcemic effect, suggest that OCIF not only inhibits the formation of osteoclasts but also affects the function and/or survival of mature osteoclasts at doses used in this study.

Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro.

The morphogenesis and remodeling of bone depends on the integrated activity of osteoblasts that form bone and osteoclasts that resorb bone. We previously reported the isolation of a new cytokine termed osteoclastogenesis inhibitory factor, OCIF, which specifically inhibits osteoclast development. Here we report the cloning of a complementary DNA of human OCIF. OCIF is identical to osteoprotegerin (OPG), a soluble member of the tumor-necrosis factor receptor family that inhibits osteoclastogenesis. Recombinant human OPG/OCIF specifically acts on bone tissues and increases bone mineral density and bone volume associated with a decrease of active osteoclast number in normal rats. Osteoblasts or bone marrow-derived stromal cells support osteoclastogenesis through cell-to-cell interactions. A single class of high affinity binding sites for OPG/OCIF appears on a mouse stromal cell line, ST2, in response to 1,25-dihydroxyvitamin D3. An anti-OPG/OCIF antibody that blocks the binding abolishes the biological activity of OPG/OCIF. When the sites are blocked with OPG/OCIF, ST2 cells fail to support osteoclastogenesis. These results suggest that the sites are involved in cell-to-cell signaling between stromal cells and osteoclast progenitors and that OPG/OCIF inhibits osteoclastogenesis by interrupting the signaling through the sites.

Bone morphogenetic protein 2 stimulates osteoclast differentiation and survival supported by receptor activator of nuclear factor-kappaB ligand.

Bone is a major storage site for TGFbeta superfamily members, including TGFbeta and bone morphogenetic proteins. It is believed that these cytokines are released from bone during bone resorption. Recent studies have shown that both RANKL and macrophage colony-stimulating factor are two essential factors produced by osteoblasts for inducing osteoclast differentiation. In the present study we examined the effects of bone morphogenetic protein-2 on osteoclast differentiation and survival supported by RANKL and/or macrophage colony-stimulating factor. Mouse bone marrow-derived macrophages differentiated into osteoclasts in the presence of RANKL and macrophage colony-stimulating factor. TGFbeta superfamily members such as bone morphogenetic protein-2, TGFbeta, and activin A markedly enhanced osteoclast differentiation induced by RANKL and macrophage colony-stimulating factor, although each cytokine alone failed to induce osteoclast differentiation in the absence of RANKL. Addition of a soluble form of bone morphogenetic protein receptor type IA to the culture markedly inhibited not only osteoclast formation induced by RANKL and bone morphogenetic protein-2, but also the basal osteoclast formation supported by RANKL alone. Either RANKL or macrophage colony-stimulating factor stimulated the survival of purified osteoclasts. Bone morphogenetic protein-2 enhanced the survival of purified osteoclasts supported by RANKL, but not by macrophage colony-stimulating factor. Both bone marrow macrophages and mature osteoclasts expressed bone morphogenetic protein-2 and bone morphogenetic protein receptor type IA mRNAs. An EMSA revealed that RANKL activated nuclear factor-kappaB in purified osteoclasts. Bone morphogenetic protein-2 alone did not activate nuclear factor-kappaB, but rather inhibited the activation of nuclear factor-kappaB induced by RANKL in purified osteoclasts. These findings suggest that bone morphogenetic protein-mediated signals cross-communicate with RANKL-mediated ones in inducing osteoclast differentiation and survival. The enhancement of RANKL-induced survival of osteoclasts by bone morphogenetic protein-2 appears unrelated to nuclear factor-kappaB activation.

Cloning and characterization of the gene encoding mouse osteoclast differentiation factor.

Osteoclast differentiation factor (ODF), a ligand for osteoclastogenesis inhibitory factor (OCIF)/ osteoprotegerin (OPG), is a member of the membrane-associated tumor necrosis factor (TNF) family and induces osteoclast-like cell formation in vitro. In the present study, mouse ODF genomic clones were isolated and sequenced to determine their gene structure. The mouse ODF gene is a single copy gene consisting of five exons and spans approximately 40kb of the mouse genome. The first exon encodes the intracellular and transmembrane domains. The extracellular region of ODF containing the TNF homologous domain is encoded by exons 1 through 5. The translation-termination codon and six polyadenylation signal residues are present in exon 5. A major transcription-initiation site is present 143 nucleotides upstream of the initiation-ATG codon. This genomic organization is similar to that of other members of the TNF family, especially the CD40 ligand.

Structure of the mouse osteoclastogenesis inhibitory factor (OCIF) gene and its expression in embryogenesis.

Osteoclastogenesis inhibitory factor (OCIF) is a novel soluble-form member of the tumor necrosis factor receptor family and is involved in the regulation of bone mass. Here we isolated genomic and cDNA clones for mouse OCIF and determined their structures. Mouse OCIF gene spans 29 kb and contains five exons of 270, 367, 192, 225 and 1765 bp long. Four cysteine-rich domains and two death domain homologous regions characterized in human OCIF are rigidly conserved in mouse OCIF. The onset of OCIF gene expression in mouse embryogenesis is at day 8.5. In a pregnant female mouse, OCIF gene is expressed in decidua, a maternal tissue surrounding each embryo, immediately after implantation. The isolation of mouse OCIF gene should facilitate studies on OCIF knock-out mice for a better understanding of the role of OCIF in vivo.

Analysis of deleted variant of hepatocyte growth factor by alanine scanning mutagenesis: identification of residues essential for its biological function and generation of mutants with enhanced mitogenic activity on rat hepatocytes.

To understand the structure-function relationship of hepatocyte growth factor (HGF) in more detail, we analyzed one of the other forms of HGF, deleted variant of HGF (dHGF), by alanine scanning mutagenesis. We show here that there are at least four sites important for dHGF to stimulate DNA synthesis in cultured adult rat hepatocytes, and that the residues of HGF essential for exerting its biological activity are not identical to those of dHGF. In addition, two mutants showed a decrease (approximately three-fold) in EC50 compared with wild-type dHGF in an assay of mitogenic activity on rat hepatocytes.

Osteoclastogenesis inhibitory factor exhibits hypocalcemic effects in normal mice and in hypercalcemic nude mice carrying tumors associated with humoral hypercalcemia of malignancy.

Osteoclastogenesis inhibitory factor (OCIF) is a novel secreted protein that inhibits osteoclastogenesis both in vitro and in vivo. In this study, we examined the effects of OCIF on serum calcium (Ca) concentrations in normal mice and in hypercalcemic nude mice carrying tumors associated with humoral hypercalcemia of malignancy. In normal mice, a single intraperitoneal injection of OCIF reduced serum Ca levels in a dose-dependent manner. Significant decrease in serum Ca (by 1.6 +/- 0.3 mg/dL, n = 5) was observed 2 h after the injection of OCIF at 20 mg/kg and the hypocalcemic effect continued for up to 12 h. Serum phosphate (Pi) concentrations also decreased in response to OCIF. Urinary excretion of Ca, Pi, and creatinine did not change significantly after injection of OCIF or vehicle. In hypercalcemic, tumor-bearing nude mice, a single intraperitoneal injection of OCIF at 20 mg/kg resulted in a dramatic decrease in serum Ca (maximal decrease 2.8 +/- 0.37 mg/dL, n = 11), which continued for up to 24 h. The results suggest that OCIF decreased serum Ca through its inhibitory effect on bone resorption. Furthermore, it is suggested that OCIF has therapeutic potential for the treatment of hypercalcemic conditions such as malignancy-associated hypercalcemia.

A novel molecular mechanism modulating osteoclast differentiation and function.

Osteoclasts, the multinucleated giant cells that resorb bone, develop from hematopoietic cells of the monocyte/ macrophage lineage. Osteoblasts, as well as bone marrow stromal cells, support osteoclast development through a mechanism of cell-to-cell interaction with osteoclast progenitors. We recently purified and molecularly cloned osteoclastogenesis inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF, a secreted member of the tumor necrosis factor (TNF) receptor family, inhibited differentiation and activation of osteoclasts. A single class of high-affinity binding sites for OPG/OCIF appeared on a mouse bone marrow stromal cell line, ST2, in response to 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] and dexamethasone (Dex). When the binding sites were occupied by OPG/OCIF, ST2 cells failed to support the osteoclast formation from spleen cells. To identify an OPG/OCIF ligand, we screened a cDNA expression library of ST2 cells treated with 1,25(OH)2D3 and Dex using OPG/OCIF as a probe. The cloned molecule was found to be a member of the membrane-associated TNF ligand family, and it induced osteoclast formation from mouse and human osteoclast progenitors in the presence of macrophage colony-stimulating factor (M-CSF) in vitro. Expression of its gene in osteoblasts/stromal cells was up-regulated by osteotropic factors, such as 1,25(OH)2D3, prostaglandin E2 (P(GE2), parathyroid hormone (PTH), and interleukin (IL)-11. A polyclonal antibody against this protein, as well as OPG/OCIF, negated not only the osteoclastogenesis induced by the protein, but also bone resorption elicited by various osteotropic factors in a fetal mouse long bone culture system. These findings led us to conclude that the protein is osteoclast differentiation factor (ODF), a long sought-after ligand that mediates an essential signal to osteoclast progenitors for their differentiation into active osteoclasts. Recent analyses of ODF receptor demonstrated that RANK, a member of the TNF receptor family, is the signaling receptor for ODF in osteoclastogenesis, and that OPG/OCIF acts as a decoy receptor for ODF to compete against RANK. The discovery of ODF, OPG/OCIF, and RANK opens a new era in the investigation of the regulation of osteoclast differentiation and function.

Osteoblasts/stromal cells stimulate osteoclast activation through expression of osteoclast differentiation factor/RANKL but not macrophage colony-stimulating factor: receptor activator of NF-kappa B ligand.

We previously reported that osteoblasts/stromal cells are essentially involved in the activation as well as differentiation of osteoclasts through a mechanism involving cell-to-cell contact between osteoblasts/stromal cells and osteoclast precursors/osteoclasts. Osteoclast differentiation factor (ODF, also called RANKL/OPGL/TRANCE) and macrophage colony-stimulating factor (M-CSF, also called CSF-1) are two essential factors produced by osteoblasts/stromal cells for osteoclastogenesis. In other words, osteoblasts/stromal cells were not necessary to generate osteoclasts from spleen cells in the presence of both ODF/RANKL and M-CSF. In the present study, we examined the precise roles of ODF/RANKL and M-CSF in the activation of osteoclasts induced by calvarial osteoblasts. Osteoclasts were formed in mouse bone marrow cultures on collagen gel-coated dishes in response to a soluble form of ODF/RANKL (sODF/sRANKL) and M-CSF, and recovered by collagenase digestion. When recovered osteoclasts were further cultured on plastic dishes, most of the osteoclasts spontaneously died within 24 h. Osteoclasts cultured for 24 h on dentine slices could not form resorption pits. Addition of sODF/sRANKL to the recovered osteoclasts markedly enhanced their survival and pit-forming activity. M-CSF similarly stimulated the survival of osteoclasts, but did not induce their pit-forming activity. When primary mouse osteoblasts were added to the recovered osteoclasts, resorption pits were formed on dentine slices. Bone-resorbing factors such as 1alpha,25-dihydroxyvitamin D3, parathyroid hormone, or prostaglandin E2 enhanced pit-forming activity of osteoclasts only in the presence of osteoblasts. M-CSF-deficient osteoblasts prepared from op/op mice similarly enhanced pit-forming activity of osteoclasts. The pit-forming activity of osteoclasts induced by sODF/sRANKL or osteoblasts was completely inhibited by simultaneous addition of osteoprotegerin/osteoclastogenesis inhibitory factor, a decoy receptor of ODF/RANKL. Primary osteoblasts constitutively expressed ODF/RANKL mRNA, and its level was upregulated by treatment with 1alpha,25-dihydroxyvitamin D3, parathyroid hormone, and prostaglandin E2. These results, obtained by using an assay system that unequivocally assesses osteoclast activation, suggest that ODF/RANKL but not M-CSF mediates osteoblast-induced pit-forming activity of osteoclasts, and that bone-resorbing factors stimulate osteoclast activation through upregulation of ODF/RANKL by osteoblasts/stromal cells.

Tumor cytotoxic activity of HGF-SF.

IMR-90 human embryonic lung fibroblasts secrete a tumor cytotoxic factor. This factor, termed F-TCF, is moderately cytotoxic in human tumor cell lines (KB, MCF-7, BG-1) and is very cytotoxic in mouse tumor cell lines (Sarcoma 180, Meth A sarcoma, P388). The cytotoxicity depends on the initial target cell number and is due to cytostasis rather than cytolysis. F-TCF was purified from conditioned medium by a combination of UF-concentration, CM sephadex C-50, Con A sepharose, Mono S cation-exchange and heparin sepharose chromatography and exhibited a molecular mass (M(r)) of 76 to 80 kD on SDS-PAGE under non-reducing conditions. F-TCF is a heterodimer composed of a large alpha-subunit with M(r) 52 to 56 kD and a small beta-subunit with M(r) 30 to 34 kD. F-TCF is a heparin-binding, heat-labile, basic glycoprotein (pI 7.4-8.6). Its activity is stable over the pH range of 6.0 to 9.0, but is completely lost after reduction with 2-mercaptoethanol. Protein sequencing indicates that the alpha-subunit is blocked at the aminoterminus. The primary amino acid sequences deduced from hepatocyte growth factor (HGF) cDNAs cloned from human placenta and liver cDNA libraries indicate that F-TCF is identical to the placenta type HGF in the aminoterminal sequence of the beta-subunit, but differs at two sites from the liver type HGF. Two forms of F-TCF cDNA were found in an IMR-90 human fibroblast cDNA library. One form was identical to placenta type HGF cDNA and the other was a variant with a 15 base pair deletion in the coding region. In addition, mRNA corresponding to the deleted form of cDNA was present in total RNA prepared from IMR-90 cells. F-TCF was thus identified as placenta type HGFs including a variant. The deleted form of recombinant HGF (rHGF) expressed in CHO cells had slightly lower heparin-binding affinity than did the intact form. Both rHGFs had almost the same dose-response curves for cytotoxicity in Sarcoma 180 or Meth A sarcoma cells. Moreover, rHGF (the deleted form) was cytotoxic in hepatocellular carcinoma cells (HepG2, Hep3B, H35). Dose-response curves for the stimulation of DNA synthesis in rat hepatocytes by HGFs were very similar up to about 12.5 ng/ml, but differed significantly at higher concentrations. The deleted form gave maximal activity in a dose range of 12.5 to 100 ng/ml and had about 1.4- to 1.9-fold higher specific activity in that range than the intact form did.(ABSTRACT TRUNCATED AT 400 WORDS)

Deleted form of hepatocyte growth factor ameliorates the mortality rate of severe thermal injury in rats.

BACKGROUND: The modulating effects of the deleted form of hepatocyte growth factor (dHGF) on burn-induced mortality rates and hepatic protein synthesis were studied in rats. METHODS: Rats were anesthetized, subjected to a 40% full-thickness scald burn, and divided into 2 groups receiving dHGF and vehicle. RESULTS: In normal rats, dHGF-treatment (1 mg/kg intravenously, twice daily) for 5 days increased the circulating plasma volume. In burned rats that were receiving vehicle, the survival rate on day 23 after the burn was 27%. The serum albumin levels were decreased and did not reverse to the normal levels until day 23 after the burn. Serum alpha 2-concentration in the injured rats was increased, whereas serum levels of transferrin, total protein, and high-density lipoprotein-cholesterol were decreased. The treatment of animals with dHGF (1 mg/kg intravenously, 3 times daily) for 3 days increased the survival rate on day 23 by 64%. In the animals treated with dHGF for 3 or 6 days, serum alpha 1-, alpha 2-, and beta-globulin concentrations were increased by the dHGF treatment. The serum levels of albumin, transferrin, total protein, and high-density lipoprotein-cholesterol reversed to normal levels or higher. CONCLUSIONS: Our data show that dHGF treatment may attenuate the decrease of the circulating plasma volume after burn and reduce a high risk of burn shock. It is also indicated that dHGF accelerates synthesis of not only acute-phase reactants but also other hepatic proteins such as albumin and transferrin on severe burn injury. These findings suggest that the appropriate upregulation of hepatic protein synthesis induced by dHGF may accelerate the physiologic recovery process after thermal injury and contribute to ameliorating the burn-induced death.

Preventive effects of the deleted form of hepatocyte growth factor against various liver injuries.

The effects of a naturally occurring deleted form of hepatocyte growth factor (HGF) on hepatic disorder were studied in various models of hepatic failure. The pretreatment of rats and mice with the deleted form of HGF prevented the liver injuries and coagulopathy induced by endotoxin, dimethylnitrosamine and acetaminophen and reduced the mortality due to hepatic dysfunction induced by these hepatotoxins. The concurrent administration of the deleted form of HGF also prevented the liver injury and hepatic fibrosis in mice treated with alpha-naphthylisothiocyanate and in rats treated with dimethylnitrosamine. Moreover, the deleted form of HGF normalized the results of the bromosulphalein-clearance test and ameliorated jaundice in rats with periportal cholangiolitic hepatopathy induced by alpha-naphthylisothiocyanate. The deleted form of HGF also reversed the coagulopathy in rats with hepatic disorder induced by dimethylnitrosamine or by 70% resection of cirrhotic liver (induced by carbon tetrachloride). In Long Evans cinnamon rats receiving vehicle, 20 out of 21 animals died within 4 days after the onset of jaundice. After infusion of the deleted form of HGF for 4 days, 7 out of 20 Long-Evans cinnamon rats survived. These results indicate that the deleted form of HGF could have therapeutic potency in patients with severe hepatic failure.