Partial characterization of a hepatocyte growth factor from rat platelets

Rat serum has been shown to stimulate DNA synthesis in primary cultures of adult rat hepatocytes 2-3 times more potently than serum from several other mammalian sources, including humans. Parallel to its stimulation of thymidine incorporation into DNA, rat serum increased the total DNA content of the hepatocyte cultures over time, and also increased the frequency of nuclear labeling and mitosis. Moreover, normal rat serum, derived from whole blood (NRS), stimulated DNA synthesis in hepatocytes twice as effectively as platelet-poor rat serum, derived from plasma (ppNRS). Addition of a rat platelet lysate (RPL) to ppNRS restored the activity to equal that of NRS. The avid binding of the active principle to CM Sephadex and its sensitivity to trypsin digestion suggest that it is a cationic polypeptide with an apparent molecular weight of about 65,000, as determined by gel filtration. It was inactivated by reduction of disulfide bonds, or by exposure to pH below 5.5, to NaCl concentration below 0.05 M, to 65 degrees C for 30 min, or to 100 degrees C for 10 min. Although it resembles the human platelet-derived mitogen platelet-derived growth factor (PDGF) in several of its properties, it differs in others. Hence the hepatocyte growth factor from rat platelets, which accounts for 50% of the DNA synthesis-stimulatory activity of rat serum, appears to be a distinct entity.     

Lung-Derived Growth Factors: Ontogenic Shift in Parahormone Secretion in the Perinatal Rat Lung

Primary cultures of perinatal rat fibroblasts were found to produce at least three mitogenic activities which exhibited specificity for distinct cell types. One activity, lung growth factor(LGF), was a potent mitogen for chick embryonal fibroblasts, which also stimulated fetal rat lung fibroblasts to undergo DNA synthesis, provided that these cells were first exposed to a "competence" factor such as fibroblast growth factor or platelet-derived growth factor. Although LGF was active in the somatomedin-C (SmC) radioimmunoassay and resembled buffalo rat liver multiplication-stimulating activity (brlMSA) in molecular size, it appears to consist of a component that is neither SmC nor brlMSA. The second activity produced by perinatal rat lung cultures, pneumocyte-stimulating activity (PSA), stimulated mitosis in type II pneumocytes of postnatal rats, and was found to have physical attributes that are distinct from those of the other known pneumocyte-influencing factors. The third activity is a non-dialyzable substance which complements the mitogenic action of LGF on fetal lung fibroblasts, and appears to be a "competence" activity. An examination of the production of LGF and PSA by rat lung fibroblasts taken at various intervals of development revealed that fetal lung fibroblasts produce maximal levels of LGF but low levels of PSA, whereas, in neonatal lung fibroblasts, the situation is reversed. This ontogenic shift in the type of parahormone produced by the developing perinatal rat lung may be an important regulatory event in postnatal lung morphogenesis in this species.     

Specificity and partial purification of a factor in spent media from Sertoli cell-enriched cultures that stimulates steroidogenesis in Leydig cells

There is increasing evidence that factors derived from the seminiferous tubules influence Leydig cell function in a paracrine way. In previous experiments we demonstrated that conditioned media from Sertoli cell-enriched cultures contain a protein with stimulatory activity on prepubertal rat Leydig cells. In this paper we further studied the specificity of this factor. In addition we describe a simple but efficient partial purification procedure. It is demonstrated that Sertoli cell conditioned media contain a factor that stimulates the testosterone output from prepubertal and adult Leydig cells. The effects are evident within the first hour of incubation and can be observed in the presence as well as in the absence of LH. Peritubular cells do not produce a similar factor but enhance the production of the Leydig cell stimulating factor when cocultured with Sertoli cells. The Sertoli cell factor acts on rat as well as on mouse Leydig cells. It barely influences the adrenostenedione output of ovarian stromal cells or the corticosterone output of adrenal cells. The production of this factor is enhanced by dbcAMP, FSH, L-isoproterenol and glucagon but is not affected by androgens. The characteristics of the Sertoli cell factor have been compared with those of a Leydig cell stimulating factor in the medium from an established rabbit kidney cell line: RK13. It is shown that the active principle in RK13 conditioned medium is also a thermolabile trypsin-sensitive protein with a mol. wt of more than 10,000. Nonetheless, the RK13 and Sertoli cell derived factors act by different mechanisms since at maximally effective concentrations their effects are additive. Finally it is demonstrated that molecular weight fractionation of Sertoli cell conditioned medium using an Amicon ultrafiltration system results in a 50- to 130-fold increase in Sertoli cell factor activity in a fraction corresponding to a mol. wt of 10,000 up to 30,000.     

Characterization of rat factors X and Xa: demonstration of factor Xa in rat plasma.

We have found that rat plasma corrected the non-activated PT of human normal or factor-X deficient plasma, and the factor Xa-like activity being constantly detected in every 1 ml of blood collected via the cannulated carotid artery of rats. The present study was undertaken to characterize the factor Xa-like activity in rat plasma by preparing rat factor X and a monoclonal antibody against it. Factor X was purified from a BaCl2 eluate of rat plasma by chromatographies on columns of DEAE-Sepharose CL-6B and Sulfate Cellulofine or on a column of Affi-Gel 10 conjugated with a monoclonal antibody against rat factor X. Factor Xa-like activity in rat plasma was eliminated by the treatment of rat plasma with a monoclonal antibody which recognized the heavy chain portions of rat factors X and Xa. A kinetical study demonstrated that rat factor Xa was strongly inhibited by rat antithrombin III, with a Ki of 2.2 x 10(-11) M, in the presence of heparin. However, in the absence of heparin, the second order rate constant for the inhibition of rat factor Xa by rat antithrombin III was 2.6 x 10(4) M-1.min-1, which was one forty-third that for the inhibition of human factor Xa by human antithrombin III. Furthermore, rat factor Xa was resistant to the inhibition by rat alpha-1-antitrypsin and alpha-2-macroglobulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological identity of a 60 kd oncofetal protein induced in rats by chemical carcinogens and released by transformed cells

A 60,000 dalton (60 kd) oncofetal protein was previously shown to be produced by tumors in tumor-bearing rats and by target tissues within 3 weeks of carcinogen treatment. The factor is released to and accumulates in the blood in vivo and in the conditioned medium of cultured transformed cells in vitro. A polyclonal antibody produced against the 60 kd factor purified from the plasma of a rat carrying the N-2-fluorenylphthalamic acid-induced transplantable Hepatoma 7777, was tested against the 60 kd factor from various sources. Based on the results of immunoprecipitation of biochemical activity associated with the 60 kd factor, it was determined that these anti-60 kd antibodies cross-reacted with the factor released by a dimethylbenzanthracene-induced rat mammary carcinoma, with the factor in rat tumor cytosol and with rat spontaneous lymphoma cells, but not with a 60 kd factor isolated from pooled cancer patient plasma. Furthermore, these antibodies cross-reacted with the 60 kd factor induced within 21 days of treatment of the rats with a range of carcinogens from 8 chemical structural groups. The anti-60 kd factor antibodies did not cross-react with a 35 kd factor having similar biochemical activity found in normal adult cells.     

Inhibitory and anti-inhibitory factors of rat serum active on the G1-S transition of hepatocyte cell cycle.

Rat hepatocytes are responsive to a serum factor inhibiting their progression through the cell cycle from the late G1 phase to the S phase. After fractionation of normal adult rat serum by two chromatographic steps on DEAE cellulose and sephadex gel filtration, the inhibitory activity was linked to proteins having a high electronegative charge and of apparent high molecular weight. Polyacrylamide gel electrophoretic analysis of active fraction showed that the alpha1 macroglobulin was its main component. Male and female baby rats were sensitive to the inhibitory factor from normal rats. Contrary to the normal adult rat serum the whole hepatectomized adult rat serum did not exhibit any ingibitory activity on the G1-S transition. However, two components having antagonist activities: an alpha1 globulin and a gamma globulin, were separated by chromatographic procedures from hepatectomized rat serum. (a) The alpha1 globulin showed an inhibitory activity. It had an apparent molecular weight lower than that found in normal rats. Its activity was sex related: only male baby rats were responsive. (b) The factor present in the gamma globulin fraction was found to be antagonistic to the alpha1 globulin factor. Its occurrence after hepatectomy explains the absence of inhibitory activity in the serum of hepatectomized rats.     

Regulation of rat lung adenylate cyclase by cytoplasmic factor(s) during development.

Basal adenylate cyclase activity in rat lung homogenate was low prenatally but increased several-fold after birth and remained elevated to maturity. The results also demonstrate the appearance of some factor(s) in the lung cytoplasm at a certain age which markedly activated adenylate cyclase. During late gestation and early neonatal life, when the cytoplasmic factor(s) was low or absent, basal adenylate cyclase activity was low and norepinephrine and NaF produced maximum activation of the enzyme. However, when the cytoplasmic factor(s) appeared in the adult lungs, basal adenylate cyclase activity was elevated and both norepinephrine and NaF produced little or no activation of the enzyme. These data suggest a role for the cytoplasmic factor(s) in regulating rat lung adenylate cyclase. The cytoplasmic factor(s) appeared to be a protein since it was inactivated by trypsin digestion and by heating to 75 degrees C. Activation of adenylate cyclase was not due to small ions or other low molecular weight components of the cytoplasm as dialysis of the supernatant did not alter its activation of adenylate cyclase. The cytoplasmic factor(s) did not appear to be either GTP or calcium-dependent regulator of cyclic AMP phosphodiesterase as these did not activate the rat lung adenylate cyclase.     

Lung-derived growth factor that stimulates the growth of lung-metastasizing tumor cells: identification as transferrin.

We have previously shown that culture medium conditioned by lung fragments contains mitogenic activity for lung-metastasizing tumor cells but not for their non-metastatic counterparts. The growth-promoting component from media conditioned by rat and porcine lungs has been purified and partially characterized as a Mr approximately 66,000 (unreduced) or Mr approximately 72,000 (reduced) glycoprotein [Cancer Res 49:3928, 1989; J Cell Biochem 43:127, 1990]. Here we report that this factor is the iron transport protein transferrin. Migration distances in sodium dodecyl sulfate and native gel polyacrylamide electrophoresis systems were similar, as were the specific activities and spectrum of mitogenic activities of the lung-derived growth factor and transferrin. Electrophoretically separated holo-rat transferrin and rat lung-derived growth factor displayed similar positive stains for iron. A polyclonal antibody generated against the lung-derived growth factor cross-reacted with human and rat transferrin in Western blots, and anti-human transferrin cross-reacted with rat lung-derived growth factor. All of the mitogenic activity contained in crude lung conditioned media could be removed by antibody-mediated transferrin depletion. The putative cell receptor molecular weights for the lung-derived growth factor and transferrin were similar as were the molecular weights of polypeptides produced by partial trypsin cleavage of the two. Finally, the amino acid sequence of certain regions of rat lung-derived growth factor demonstrated a high degree of homology to human transferrin. The physical and biochemical properties, antigenicity, and mitogenic activity of a previously unidentified lung-derived growth factor for lung-metastasizing tumor cells indicate that it is transferrin.     

Properties of a growth factor activity present in crude extracts of rat uterus

We have shown previously (D.A. Sirbasku, 1978, Proc. Natl. Acad. Sci. U.S.A., 75:3786–3790) that an estrogen-inducible growth factor activity for rat mammary and rat pituitary tumor cells can be identified in extracts of rat uteri, although at the time of that report only a limited biochemical characterization of the activity was presented. In this report, we have evaluated the growth factor activity for lipid, steroid hormone or protein-like properties. Uterine growth factor activity was assayed by measure of the increased cell number of the MTW9/PL rat mammary tumor cell line established by this laboratory and described previously (D.A. Sirbasku, 1978, Cancer Res. 38:1154–1165). Studies showed the following characteristics of growth factor activity: destroyed by trypsin treatment; labile when heated at 80°C; partially denatured by 6 M guanidine or 8 M urea treatment or 50% aqueous solutions of organic solvents; inactivated by extremes of pH or overnight treatment with mild acid; not dialyzable at neutral pH; of apparent molecular weight of 70,000 daltons by G-100 Sephadex chromatography; possessing an isoelectric point of 4.8 to 5.2; not chloroform/methanol extractable; and not in any way identified as either a lipid or a steroid hormone. The data available suggest that the uterine growth factor activity is a protein or polypeptide of apparent high molecular weight, and that this activity does not directly correspond to other known growth factors.     

Rat Serum Factors Inhibiting the G1-S Transition In Hepatocytes

An attempt was made to detect the serum factors inhibiting the G₁-S transition in synchronized, baby rat hepatocytes. In untreated adult rat serum, this inhibitory activity was always linked to high molecular weight (HMW) compounds. Incubation of serum with trypsin or chymotrypsin resulted in the formation of a low molecular weight (LMW) G₁-S inhibitory factor. the same result was obtained with fractions from adult rat liver but not with kidney or spleen fractions. Separation of the LMW factor by ultrafiltration increased its specific activity by about 10³. the active period in the cell cycle of both the LMW and HMW factors was the same: the late G₁ phase. However, the activity of the LMW factor was not blocked by the Kunitz factor. an enzymatic transformation of the HMW factor might be induced by liver cell membrane-bound proteases and constitute a mechanism regulating hepatocyte proliferation.     

Factors in the rat submaxillary gland that stimulate growth of cultured glioma cells: identification and partial characterization

The effect of rat submaxillary extract on the growth of rat C6 glioma cells in serum-free culture has been examined. Extracts (10-15 microgram/ml) of submaxillary glands from both male and female rats markedly enhanced the growth of serum-deprived C6 cells and, in combination with insulin, transferrin, and NIH-LH (a source of fibroblast growth factor), were able to stimulate C6 cell growth to an extent comparable to that achieved with an optimal amount of fetal calf serum. The mitogenic activity of rat submaxillary extracts was found to be heat-labile, acid-stable, and partially inactivated by protease and 2-mercaptoethanol. Under our assay conditions, biologically active preparations of purified mouse submaxillary gland epidermal growth factor (EGF) or nerve growth factor (NGF) were not mitogenic for C6 cells, nor was the mitogenic activity of rat submaxillary extracts inhibited by antiserum to these mouse submaxillary gland growth factors. These results suggest that the active component(s) of rat submaxillary extracts is unrelated to either EGF or NGF. The growth-enhancing effect also appears unrelated to esteropeptidase activity present in these extracts since the mitogenic activity was unaffected by several protease inhibitors. Moreover, two purified mouse submaxillary gland arginylesteropeptidases, EGF-binding protein and gamma-subunit of 7 S NGF, were unable to elicit a comparable growth response even when added to cell culture medium at unreasonably high concentrations. The C6 cell mitogenic activity of crude submaxillary extracts could be separated into two biologically similar components by either gel filtration on Sephadex G-100, preparative isoelectric focusing in a pH gradient of 3-10, or adsorption to DEAE-cellulose followed by elution with a sodium chloride gradient. One of the active components was acidic in nature and had an apparent molecular weight of 40,000, while the other was near neutral in charge and possessed a molecular weight of approximately 20,000. The relationship between these two C6 cell mitogenic components and the rat submaxillary gland component responsible for stimulating Balb/c-3T3 cell growth in serum-free, factor supplemented medium (McClure et al., 1979, J. Cell Biol. 83:96a) is also discussed.     

Regulation of rat lung adenylate cyclase by cytoplasmic factor(s) during development

Basal adenylate cyclase activity in rat lung homogenate was low prenatally but increased several-fold after birth and remained elevated to maturity. The results also demostrate the appearance of some factors(s) in the lung cytoplasm at a certain age which markedly activated adenylate cyclase. During late gestation and early neonatal life, when the cytoplasmic factor(s) was low or absent, basal adenylate cyclase activity was low and norepinephrine and NaF produced maximum activation of the enzyme. However, when the cytoplasmic factor(s) appeared in the adult lungs, basal adenylate cyclase activity was elevated and both norepinephrine and NaF produced little or no activation of the enzyme. These data suggest a role for the cytoplasmic factor(s) in regulating rat lung adenylate cyclase.The cytoplasmic factor(s) appeared to be a protein since it was inactivated by trypsin digestion and by heating to 75°C. Activation of adenylate cyclase was not due to small ions or other low molecular weight components of the cytoplasm as dialysis of the supernatant did not alter its activation of adenylate cyclase. The cytoplasmic factor(s) did not appear to be either GTP or calcium-dependent regulator of cyclic AMP phosphodiesterase as these did not activate the rat lung adenylate cyclase.     

Factor Xa induces mitogenesis of vascular smooth muscle cells via autocrine production of epiregulin

Factor Xa has been reported to elicit smooth muscle cell proliferation via autocrine release of platelet-derived growth factor. However, this study has shown that factor Xa-induced mitogenesis of rat aortic smooth muscle cell is independent of platelet-derived growth factor. We also could not observe any platelet-derived growth factor isoforms in the cultured medium of factor Xa-stimulated cells. Our finding that the cultured medium of factor Xa-stimulated cells strongly induces rat aortic smooth muscle cell mitogenesis in the absence of factor Xa activity led us to explore the existence of a novel autocrine pathway. The autocrine growth factor was purified from the cultured medium and was identified to be epiregulin. Recombinant epiregulin was also able to induce the mitogenesis. The secretion of epiregulin from factor Xa-stimulated rat aortic smooth muscle cell required mRNA expression and protein synthesis of the growth factor. The mitogenic effect of factor Xa on rat aortic smooth muscle cell was significantly reduced by anti-epiregulin antibody or by antisense oligodeoxynucleotide to epiregulin. Several lines of experimental evidence clearly indicate that the autocrine production of epiregulin, an epidermal growth factor-related ligand, is induced in the factor Xa-stimulated mitogenic process of rat aortic smooth muscle cell.     

Differentiation of intestinal epithelial cell line (IEC-18) by an acid extract of rat small intestine

A factor which may induce differentiation of intestinal epithelial cell lines in vitro was found in an acid extract of adult rat small intestine. The addition of a partially purified acetic acid extract of rat small intestine to IEC-18 cell culture dishes increased sucrase activity within 48 h. Thymidine incorporation markedly decreased within 24 h. Significant development of microvilli-like structures was observed on the acid extract-treated IEC-18 cells, compared with controls. This activity of rat acid extract was heat-stable and the apparent molecular weight of the factor was 400-800. These findings suggested that the factor may be related to the epithelial differentiation of rat small intestinal crypt cells.     

Phosphorylation of supernatant protein factor enhances its ability to stimulate microsomal squalene monooxygenase

Supernatant protein factor is a 46-kDa cytosolic protein that stimulates squalene monooxygenase, a downstream enzyme in the cholesterol biosynthetic pathway. The mechanism of stimulation is poorly understood, although supernatant protein factor belongs to a family of lipid-binding proteins that includes Sec14p and alpha-tocopherol transfer protein. Because recombinant human supernatant protein factor purified from Escherichia coli exhibited a relatively weak ability to activate microsomal squalene monooxygenase, we investigated the possibility that cofactors or post-translational modifications were necessary for full activity. Addition of ATP to rat liver cytosol increased supernatant protein factor activity by more than 2-fold and could be prevented by the addition of inhibitors of protein kinases A and C. Incubation of purified recombinant supernatant protein factor with ATP and protein kinases A or C delta similarly increased activity by more than 2-fold. Addition of protein phosphatase 1 gamma, a serine/threonine phosphatase, to rat liver cytosol reduced activity by 50%, suggesting that supernatant protein factor is partially phosphorylated in vivo. To determine whether dietary cholesterol influenced the phosphorylation state, cytosols were prepared from livers of rats fed a high fat diet. Although supernatant protein factor activity was reduced by more than one-half, it could not be restored by the addition of ATP or protein kinase C delta with ATP, suggesting that dietary cholesterol reduced the expression of this protein. Supernatant protein factor thus appears to be regulated both post-translationally through phosphorylation and at the level of expression. Phosphorylation may provide a means for the rapid short term modulation of cholesterol synthesis.     

A homologue of platelet-derived growth factor produced by rat alveolar macrophages

Rat alveolar macrophages secrete a growth factor that renders rat lung fibroblasts competent to initiate DNA synthesis in vitro in the presence of platelet-poor plasma. This biological activity resembles that of platelet-derived growth factor (PDGF). After separation from putative associated binding proteins by chromatography under acidic conditions, the macrophage-derived factor exhibited a relative molecular weight similar to that of highly purified human PDGF. The factor bound to a monospecific antibody to human PDGF and thus could be quantitated in an enzyme immunoassay for PDGF. It competed with radiolabeled human PDGF for receptor sites for PDGF on rat lung fibroblasts, and binding to these receptor sites could be specifically inhibited by anti-PDGF. These data strongly support the view that the factor derived from rat alveolar macrophages is homologous to human PDGF and is similar to human macrophage-derived PDGF-like growth factor. Furthermore, we have demonstrated that the lung contains both an effector cell (pulmonary macrophage) and a potential target cell (interstitial fibroblast) for this cytokine. Therefore the rat appears to be an appropriate animal model in which to study macrophage-derived PDGF-like growth factors as mediators of proliferation in pulmonary fibrogenesis.     

Purification of a skeletal muscle polypeptide which stimulates choline acetyltransferase activity in cultured spinal cord neurons

Extracts of rat skeletal muscle contain neurotrophic factors which stimulate the development of choline acetyltransferase in embryonic day 14 rat spinal cord cultures. The trophic activity does not bind heparin-Sepharose or lectin affinity columns. However, mild acid treatment separates the trophic activity into soluble and insoluble fractions. The acid-insoluble activity has been purified 5000-fold to apparent homogeneity using preparative sodium dodecyl sulfate gel electrophoresis to achieve final purification. The purified factor migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, with an apparent molecular mass of 20 kDa and a pI of 4.8. The activity and apparent molecular weight of the purified factor are unaltered by treatment with reducing agents or incubation in acidic conditions. Activity, however, is destroyed by heating or protease treatment. Thus, the factor appears to be a single polypeptide without significant levels of glycosylation or charge microheterogeneity. These results represent the first purification of a neurotrophic factor from skeletal muscle. The physical properties and amino acid composition of this factor differ from those of nerve growth factor and heparin-binding growth factors, as well as from the neurotrophic factor from heart cell conditioned medium which induces cholinergic development in sympathetic neurons.     

Platelet derived growth factor(s) for a hormone-responsive rat mammary tumor cell line.

Mammary tumor cell growth factor(s) has been identified in extracts of platelets from both male and female rats, as well as in extracts prepared from pooled outdated human platelets. When assayed by the growth promotion of MTW9/PL rat mammary tumor cells in culture, platelet extracts alone were able to support growth 50--75% as well as whole serum. The mitogenic activity from crude human platelet lysates was shown to be trypsin sensitive, relatively stable to extremes of pH, labile to heat treatment at 70 degrees, non-dialysable, ammonium sulfate precipitable, not removed by 56 degrees charcoal treatment, and of apparent molecular weight of 30,000 to 50,000 daltons as estimated by G-100 Sephadex chromatography. The platelet derived mammary growth factor activity was not replaced or potentiated by thrombin or known hormones and growth factors such as prolactin, insulin, 17-beta-estradiol, progesterone, hydrocortisone, L-thyroxine, and mouse epidermal growth factor. The experimental report demonstrates that platelets are a rich source growth factor activity for rat epithelial mammary tumor cells, and that the activity appears to be a polypeptide(s) different from other mitogenic activities known to influence growth of mammary tissue.     

Cardiac lipoprotein lipase: effects of lipopolysaccharide and tumor necrosis factor

Summary Lipopolysaccharide (LPS), the active principle of certain endotoxins, protein-free perfused in rat hearts leads in 3 h to a considerable loss of lipoprotein lipase (LPL) activity. In the presence of albumin LPS has virtually no effect. Tumor necrosis factor (TNF) added instead of LPS had no effects on LPL activity during 3 hin vitro perfusion. LPS injected into rats intravenously leads within 3 h to severe toxic phenomena amongst which increased capillary permeability. This was visualized as increased rate of interstitial fluid formation in Langendorff hearts mounted 3 h after rats had been treated with LPS. LPL activity did not decline in 3 h lasting endotoxemia. Six hours after LPS injection, however, cardiac LPL activity was considerably lowered, although immunoblotting and immunohistochemistry still showed LPL protein to be present. These date indicate the presence of a considerable pool of inactive LPL protein in addition to active LPL, that can be released in the presence of heparin. The LPL activity is lowered by LPS injection after a lag phase of at least 3 h, while capillary endothelial cells are influenced more rapidly. The relatively late expression of TNF toxicity in cardiomyocytes of the intact heart is discussed.