Heparin-binding epidermal growth factor-like growth factor (HB-EGF) and proteolytic processing by a disintegrin and metalloproteinases (ADAM): a regulator of several pathways.

HB-EGF is a member of the EGF family of ligands that is initially synthesized as a membrane-bound growth factor termed, proHB-EGF. The membrane bound proHB-EGF undergoes extensive proteolytic processing by several metalloproteinases capable of stimulating cellular proliferation. Soluble, mature HB-EGF binds to and activates EGF receptors. HB-EGF is a critical molecular component to a number of normal physiological processes including but not limited to tissue injury and wound healing, reproduction, angiogenesis and recently, adipogenesis. Misexpression of HB-EGF is linked to tumor formation and cancer including hepatocellular, pancreatic, gastric, breast, colon and melanoma, gliomas and glioblastomas. HB-EGF is a likely tool for therapeutic approaches to enhance treatment of injuries as well as a target for prevention of several cancers and obesity.

Cellular reprogramming into a brown adipose tissue-like phenotype by co-expression of HB-EGF and ADAM 12S.

Abnormal adipogenesis leads to excessive fat accumulation and several health disorders. Mouse fibroblasts (MLC) transfected with ADAM 12S and HB-EGF promoted lipid accumulation. Addition of KBR-7785, an ADAM 12S inhibitor, to HB-EGF/ADAM 12S expressing cells suppressed adipogenesis. BrdU incorporation was attenuated and enhanced mitotracker staining was observed in HB-EGF/ADAM 12S cells. Quantitative real time RT-PCR resulted in elevated levels of expression of three brown adipose tissue (BAT) genes (PRDM16, PGC-1α, and UCP-1), while expression levels of the three white adipose tissue (WAT) genes (PPARγ, C/EBPα, and AKT-1) were unaltered in HB-EGF/ADAM 12S cells. Amino- or carboxy-terminal deletions of HB-EGF (HB-EGFΔN and HB-EGFΔC) co-expressed with ADAM 12S stimulated lipid accumulation. Human epidermoid carcinoma cells (A431) also exhibited lipid accumulation by HB-EGF/ADAM 12S co-expression. These studies suggest ADAM 12S and HB-EGF are involved in cellular plasticity resulting in the production of BAT-like cells and offers insight into novel therapeutic approaches for fighting obesity.

Polymorphism at the avpr1a locus in male prairie voles correlated with genetic but not social monogamy in field populations.

Integrative studies of genetics, neurobiology and behaviour indicate that polymorphism in specific genes contributes to variation observed in some complex social behaviours. The neuropeptide arginine vasopressin plays an important role in the regulation of a variety of social behaviours, including social attachment of males to females, through its action on the vasopressin 1a receptor (V1aR). In socially monogamous prairie voles (Microtus ochrogaster), polymorphism in the length of microsatellite DNA within the regulatory region of the gene (avpr1a) encoding the V1aR predicts differences among males in neural expression of V1aRs and partner preference under laboratory conditions. However, understanding the extent to which V1aR mediates variation in prairie vole social and reproductive behaviour observed in nature requires investigating the consequences of avpr1a polymorphism and environmental influences under ecologically relevant conditions. We examined the relationship between avpr1a length polymorphism and monogamy among male prairie voles living in 0.1 ha enclosures during a time similar to their natural lifespan. We found no evidence that avpr1a genotype of males predicts variation in social monogamy measured in the field but some indices of social monogamy were affected by population density. Parentage data indicated that a male's avpr1a genotype significantly influenced the number of females with which he sired offspring and the total number of offspring sired. Total brain concentrations of V1aR mRNA were not associated with either male behaviour or avpr1a genotype. These data show that melding ecological field studies with neurogenetics can substantially augment our understanding of the effects of genes and environment on social behaviours.

The significance of disulfide bonding in biological activity of HB-EGF, a mutagenesis approach.

A site-directed mutagenesis approach was taken to disrupt each of 3 disulfide bonds within human HB-EGF by substituting serine for both cysteine residues that contribute to disulfide bonding. Each HB-EGF disulfide analogue (HB-EGF-Cys/Ser(108/121), HB-EGF-Cys/Ser(116/132), and HB-EGF-Cys/Ser(134/143)) was cloned under the regulation of the mouse metallothionein (MT) promoter and stably expressed in mouse fibroblasts. HB-EGF immunoreactive proteins with M(r) of 6.5, 21 and 24 kDa were observed from lysates of HB-EGF and each HB-EGF disulfide analogue. HB-EGF immunohistochemical analyses of each HB-EGF stable cell line demonstrated ubiquitous protein expression except HB-EGF-Cys/Ser(108/121) and HB-EGF-Cys/Ser(116/132) stable cell lines which exhibited accumulated expression immediately outside the nucleus. rHB-EGF, HB-EGF, and HB-EGF(134/143) proteins competed with 125I-EGF in an A431 competitive binding assay, whereas HB-EGF-Cys/Ser(108/121) and HB-EGF-Cys/Ser(116/132) failed to compete. Each HB-EGF disulfide analogue lacked the ability to stimulate tyrosine phosphorylation of the 170 kDa EGFR. These results suggest that HB-EGF-Cys/Ser(134/143) antagonizes EGFRs.

Amino-terminal deletion of heparin-binding epidermal growth factor-like growth factor4-127 stimulates cell proliferation but lacks insulin-like activity.

UNLABELLED: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) Northern analysis demonstrated a novel 0.8-kb liver-specific HB-EGF transcript in addition to the endogenous 2.5-kb HB-EGF full-length transcript present in kidney, lung and liver tissues. Reverse transcriptase-polymerase chain reaction screening of liver RNA suggests that the 0.8-kb HB-EGF transcript lacks at least a portion of the amino-terminal EGF-like domain. In light of these data, we have constructed a human HB-EGF cDNA (HB-EGF(DeltaN)) which lacks 373 bp, encoding the majority of the extracellular EGF-like domain, while maintaining the ectodomain 'shedding' site, transmembrane and cytoplasmic domains. OBJECTIVE: The goal of this study is to characterize the ability of HB-EGF(DeltaN) to (i) stimulate cell proliferation and (ii) determine whether down-regulation of insulin-like growth factor-binding protein (IGFBP)-3 and -4 mRNA is regulated by soluble, mature HB-EGF or HB-EGF C. MATERIALS AND METHODS: HB-EGF(DeltaN) encodes nucleotides +1-10 of exon 1 linked to nucleotides 383-627 of the carboxy-terminal portion of exon 3 through exon 5. RESULTS: Expression of HB-EGF(DeltaN) in mouse fibroblasts (MLC) resulted in 6.5- and 8-kDa HB-EGF immunoreactive proteins, stimulated tyrosine phosphorylation of p42 kDa and cell proliferation in MLC, but lacked the ability to bind EGF receptors. Finally, HB-EGF(DeltaN) failed to down-regulate IGFBP-3 and -4 mRNA when expressed in normal rat kidney cells. CONCLUSIONS: These findings demonstrate that amino-terminally truncated, membrane-bound form of HB-EGF stimulates cell proliferation but lacks insulin-like signalling, suggesting that insulin-like signalling is mediated by soluble, mature HB-EGF binding to EGF receptors.

Characterization of the gene encoding murine heparin-binding epidermal growth factor-like growth factor.

The mouse gene (mHB-EGF) encoding heparin-binding epidermal growth factor-like growth factor was isolated from a mouse 129SVJ genomic library. DNA sequence analysis confirmed that the clone contained six exons (I-VI) and five introns (A-E), and spanned approx. 14 kb of DNA. PCR analysis showed that introns A-E of mHB-EGF are 203 bp, 2.5 kb, 5.5 kb, 825 bp and 272 bp in length, respectively. These results establish that mHB-EGF is similar in organization to human HB-EGF (hHB-EGF). However, DNA sequence analysis of introns A-E of mHB-EGF failed to show significant overall homology with those of hHB-EGF.

Expression of a functional porcine growth hormone receptor cDNA in mouse L cells.

Porcine (p) growth hormone receptor (GHR) complementary DNA (cDNA) has been cloned and the primary amino acid structure was deduced from the nucleotide sequence. A comparison of pGHR to other GHRs revealed an approximately 70% similarity in amino acid sequence (Cioffi et al., 1990). Hybridization of this receptor cDNA to RNA samples isolated from various porcine tissues revealed a single RNA band of 4.2 kb. The full-length pGHR cDNA was subcloned into an eukaryotic expression vector, transcription of which was directed by the mouse metallothionein-I transcriptional regulatory sequence. Stable mouse L cell lines which express the pGHR cDNA were established. Approximately 80% of the cell lines were found to possess pGHR mRNA (approximately 2 kb) which corresponds to the length of the cloned pGHR cDNA. Binding studies showed that the stable cell lines were capable of specifically binding 125I-labeled pGH with a dissociation constant (Kd) of approximately 1.0 nM. The apparent molecular mass of the receptor, as determined by cross-linking studies, was found to be 118 kDa. Also, the receptor-ligand complex could be internalized. These results suggest that an active form of pGHR had been cloned and stably expressed in mouse L cells.

In vitro mutagenesis of growth hormone receptor Asn-linked glycosylation sites.

Site-directed mutagenesis was used to replace asparagine (Asn) residues with glutamine (Gln) at the five potential N-linked glycosylation sites located at positions 28, 97, 138, 143, and 182 in the extracellular domain of the porcine growth hormone receptor (pGHR). These mutated pGHR cDNAs were stably expressed in mouse L cells. Single substitution of the Asn residues did not alter growth hormone binding when compared to cells which express native pGHR (KD approximately 1 nM). However, substitution of the five potential Asn-linked sites together (pGHR delta 5) resulted in a 20-fold reduced GH binding affinity (KD = 20 nM). Residues Asn97, Asn138, and Asn182 were apparently glycosylated and upon cross-linking with 125I-labeled pGH migrated as a molecular complex of approximately 130 kDa. Native pGHR and pGHR analogs with substitutions of N28Q and N143Q when cross-linked to 125I-labeled pGH, migrated with a Mr of 138 kDa. The fully deglycosylated cross-linked receptor, pGHR delta 5, migrated as a complex of 108 kDa. Therefore, each carbohydrate moiety contributed approximately 10 kDa to the total molecular mass of the pGHR, in sum contributing 30 kDa to the total Mr of the glycosylated pGHR. pGHR delta 5 was able to internalize nearly all the bound 125I-labeled pGH within 10 min, whereas native pGHR and individual Asn substituted pGHR analogs internalized 25% of bound 125I-labeled pGH at 10 min. Also, mutagenesis of the pGHR five potential Asn-linked glycosylation sites, either singly or together, did not alter the ability of GH to induce tyrosine phosphorylation of a 95-kDa protein. Together, the results indicate that three of the five pGHR Asn residues are apparently glycosylated and are necessary for maintenance of a high affinity GH binding site and for GH internalization. However, glycosylation of the pGHR is not critical for eliciting tyrosine phosphorylated proteins following the GH/GHR interaction.

Characterization of pig connective tissue growth factor (CTGF) cDNA, mRNA and protein from uterine tissue.

Connective tissue growth factor (CTGF) is a 38kDa mitogen and chemotactic factor for fibroblasts that is transcriptionally activated by serum or transforming growth factor-beta and may play a role in wound healing and various skin diseases. In these studies, pig endometrium was shown to contain a single CTGF transcript of 2.4kb and to produce a 38kDa CTGF-immunoreactive protein. Cloning and sequencing of a 1.5kb pig uterine CTGF cDNA revealed that the predicted pCTGF primary translation product displayed 92% identity to human CTGF and 93% identity to mouse CTGF. The pCTGF cDNA encoded a 26 amino acid signal peptide followed by a 323-residue sequence containing 38 highly conserved cysteine residues. In common with mouse and human CTGF proteins, pCTGF is predicted to resemble a multi-functional mosaic protein that contains four distinct modules.

Cloning of a pig glucose transporter 4 cDNA fragment: use in developing a sensitive ribonuclease protection assay for quantifying low-abundance glucose transporter 4 mRNA in porcine adipose tissue.

A 246-bp fragment of porcine glucose transporter 4 (GLUT4) cDNA was cloned by polymerase chain reaction (PCR) from porcine adipose tissue RNA. Nucleotide sequences 1-138 and 139-246 of the GLUT4 cDNA share 78% sequence identity with exon 4a and 91% sequence identity with exon 4b of the human GLUT4 gene, respectively. The GLUT4 cDNA fragment was subcloned into pGEM-4Z vector to synthesize a highly specific riboprobe that hybridized only to human GLUT4 cDNA but not to human glucose transporter 1 (GLUT1) cDNA. Northern blot analysis of total RNA revealed the presence of a single transcript of 2.8 kb in porcine adipose tissue. Cloning a fragment of the GLUT4 cDNA enabled us to develop a ribonuclease protection assay for detecting porcine GLUT4 mRNA. The ribonuclease (RNase) protection assay is highly reproducible and retains a sensitivity level to as little as 2 pg of GLUT4 mRNA. The standard curve was linear between 2 and 128 pg of sense-strand GLUT4 RNA (r = .994). The ability to detect small quantities of GLUT4 mRNA is important when the abundance of GLUT4 mRNA is low and the quantity of tissue is limiting (e.g., when RNA is extracted from cultured adipose tissue). When porcine adipose tissue explants were cultured in the presence of insulin (10 ng/mL), GLUT4 mRNA abundance was increased. Development of a sensitive assay to quantify GLUT4 mRNA in porcine adipose tissue will enable us to conduct studies to increase our understanding of the molecular mechanisms by which porcine somatotropin (pST) regulates GLUT4 gene expression.

Growth hormone (GH)-induced tyrosine-phosphorylated proteins in cells that express GH receptors.

We have shown previously that growth hormone (GH)-induced tyrosine phosphorylation of a 95-kDa protein in mouse L-cells stably transfected with the GH receptor. In addition to induction of pp95, we have established that GH also induces tyrosine phosphorylation of a 42-kDa protein and a 130-kDa protein, as detected with phosphotyrosine antibodies. A time course of tyrosine phosphorylation on GH treatment indicates that within the GH signal transduction cascade, tyrosine phosphorylation of pp95 occurs by 1 min, whereas tyrosine phosphorylation of pp42 was not detected until 5 min. Additionally, the concentration of GH needed to stimulate tyrosine phosphorylation of pp42 was greater than that required for pp95. The pp42 protein comigrates with a 42-kDa protein identified as extracellular signal-regulated kinase 2 (ERK2). Growth factors, such as FGF, PDGF, IGF-I, and insulin, induce tyrosine phosphorylation of pp42 in pGHR-W10 cells and in 3T3-F442A preadipocytes; however, they are unable to induce pp95. These results suggest that GH induction of tyrosine-phosphorylated pp42 may represent a common signal transduction point of various growth factors, including GH, whereas tyrosine phosphorylation of pp95 is GH specific.

A comparison of cholestyramine and nicotinic acid in the treatment of familial type II hyperlipoproteinaemia.

1 The effects of cholestyramine and nicotinic acid on plasma lipid concentration have been compared in patients with type IIa hyperlipoproteinaemia. 2 During a 3-month period, cholestyramine resulted in a mean decrease in cholesterol levels of 26%. Triglyceride levels rose in eight of the ten patients during treatment with this drug but in the majority of patients remained within the normal range. 3 During nicotinic acid therapy, cholesterol fell by a mean of 21% and triglyceride by a mean of 23%. 4 The slow release preparation of nicotinic acid used was acceptable to the majority of the patients studied and the results therefore suggest that this drug may be a useful alternative to the more widely used agent, cholestyramine.

In vitro effects of IL-12 on HIV-1-specific CTL lines from HIV-1-infected children.

We studied the in vitro effects of IL-12 on HIV-1-specific CTL lines derived from PBMC of HIV-1-infected children. HIV-1-specific CTL lines were derived by limiting dilution following Ag-specific stimulation of PBMC from HIV-1-infected children and were maintained with repeated anti-CD3 stimulation. Following incubation with IL-12 for 5 to 7 days, HIV-1-specific cytotoxicity was augmented in a dose-dependent fashion (mean increase, 94 +/- 83 lytic units; p = 0.0006). Experiments performed with CD3-blocking Abs and MHC-mismatched targets demonstrated that the IL-12-enhanced activity was MHC restricted and dependent on cells bearing CD3. The effect of IL-12 on proliferation of the CTL lines as tested by [3H]TdR uptake was minimal, with stimulation indexes ranging from 1.25 to 4.9. The effects of IL-12 on cytotoxicity were not significantly altered by addition of Ab to the IL-2R (anti-Tac) in quantities sufficient to block exogenous IL-2 (p = 0.15), demonstrating that endogenous IL-2 activity is not required for IL-12-enhanced cytolytic activity. Likewise, addition of neutralizing Ab specific for IFN-gamma did not change IL-12-enhanced cytotoxicity (p = 0.61). The in vivo role of IL-12 in the generation and the stimulation of CTL remains to be determined; however, its ability to augment HIV-1-specific CTL in vitro adds additional support for IL-12 as a candidate for immune-based therapy of HIV-1.

High frequency of Gag- and envelope-specific cytotoxic T lymphocyte precursors in children with vertically acquired human immunodeficiency virus type 1 infection.

Circulating human immunodeficiency virus type 1 (HIV-1)-specific cytotoxic T lymphocytes (CTL) are seen less frequently in unstimulated peripheral blood mononuclear cells (PBMC) from children with vertically acquired HIV infection than in PBMC from HIV-infected adults. HIV-1 Gag-, reverse transcriptase (RT)-, and envelope (Env)-specific cytotoxic activity was studied in PBMC from HIV-infected children. Only 9% of subjects had Gag- or RT-specific CTL in unstimulated PBMC. However, in PBMC studied after CD3 stimulation, Gag- and Env-specific CTL were found in PBMC from 91% and 78% of HIV-infected children, respectively. Limiting dilution analysis of precursor CTL (pCTL) frequencies in PBMC from children > 12 months old demonstrated Gag- and Env-specific pCTL frequencies from 0.5 to 6.3/10,000 PBMC and from 0.66 to 33.0/10,000 PBMC, respectively. Thus, children with vertically acquired HIV infection have high frequencies of HIV-specific pCTL.

Growth hormone (GH) and a GH antagonist promote GH receptor dimerization and internalization.

It has previously been shown that a human growth hormone (hGH) analog, hGH-G120R, acts as a GH antagonist (Chen, W. Y., Wight, D. C. , Wagner, T. E., and Kopchick, J. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5061-5065; Chen, W. Y., White, M. E., Wagner, T. E., and Kopchick, J. J. (1991) Endocrinology 129, 1402-1408; Chen, W. Y., Chen, N-Y., Yun, J., Wang, X. Z., Wagner, T. E., and Kopchick, J. J. (1994) J. Biol. Chem. 269, 15892-15897). In this study, we report the ability of hGH and hGH-G120R to be internalized by GH receptor expressing cells. Additionally, results of chemical cross-linking experiments revealed that both native hGH and hGH-G120R form complexes similar in size to that expected for hGH when bound to recombinant hGH-binding protein (bp). The molecular mass of the complex was determined to be approximately 280 kDa which is consistent with multiple receptors interacting with the ligand. The predominant radiolabeled band detected was a complex of approximately 140 kDa which probably represents one GH molecule bound to one GH receptor. The cross-linked complexes were not detected in the presence of excess unlabeled hGH or hGH-G120R and were not observed in cells which do not express detectable levels of GH receptors. Also, GH induced tyrosine phosphorylation of a complex of proteins of approximately 95 kDa in these cells whereas hGH-G120R did not. Thus, we have separated the hGH or hGH-G120R/GHR binding and internalization capabilities from the ability to stimulate tyrosine phosphorylation of intracellular proteins.

Characterization of cell-associated and soluble forms of connective tissue growth factor (CTGF) produced by fibroblast cells in vitro.

Connective tissue growth factor (CTGF) is a mitogenic and chemotactic factor for cultured fibroblasts that has been implicated in wound healing, fibrotic disorders and uterine function. Although the primary translational products of the mouse, human and pig CTGF (mCTGF, hCTGF, pCTGF) genes are predicted to be secreted and of approximate M(r) 38,000, 10 kDa biologically active forms of pCTGF have recently been described. In this report, we show that human foreskin fibroblasts (HFFs) and mouse connective tissue fibroblasts contained 2.4 kb CTGF transcripts, stained positively with an anti-CTGF[81-94] peptide antiserum, and produced a 38 kDa protein that was immunoprecipitated by an anti-CTGF[247-260] peptide antiserum. While 38 kDa CTGF was readily detected in cell lysates, it was non- or barely detectable in conditioned medium. 38 kDa CTGF remained cell-associated for at least 5 days after synthesis and was not releasable by treatment of the cells with trypsin, heparin, 1 M NaCl or low pH. Purification of CTGF from human or mouse fibroblast conditioned medium resulted in the isolation of 10-12 kDa CTGF proteins that were heparin-binding, bioactive, and reactive with anti-CTGF[247-260] on Western blots. Whereas 10 kDa CTGF stimulated DNA synthesis in 3T3 cells to the same extent as platelet-derived growth factor (PDGF)-AA, -AB, or -BB, it did not compete with 125I-PDGF-BB for binding to alpha alpha, alpha beta or beta beta PDGF receptors (PDGF-R), did not stimulate tyrosine phosphorylation of PDGF-alpha-R or -beta-R, and was not antagonized by a neutralizing PDGF-R-alpha antiserum. These data show that, in cultured fibroblasts, 38 kDa CTGF is principally cell-associated whereas low mass forms of CTGF are soluble and biologically active. They further demonstrate that, contrary to the previously proposed properties of 38 kDa CTGF, 10 kDa CTGF does not bind to PDGF-R and stimulates Balb/c 3T3 cell mitosis via a PDGF-R-independent mechanism.

Purification and characterization of novel heparin-binding growth factors in uterine secretory fluids. Identification as heparin-regulated Mr 10,000 forms of connective tissue growth factor.

Uterine growth factors are potential effector molecules in embryo growth signaling pathways. Pig uterine luminal flushings contained a heparin-binding growth factor (HBGF) that required 0.8 M NaCl for elution from heparin columns and was termed HBGF-0.8. This factor, which was heat- and acid-labile and of Mr 10,000 as assessed by gel filtration, stimulated DNA synthesis in fibroblasts and smooth muscle cells but not endothelial cells. Two forms of HBGF-0.8, termed HBGF-0.8-P1 and HBGF-0.8-P2, exhibited differential heparin-binding properties. SDS-polyacrylamide gel electrophoresis showed that each form of HBGF-0.8 migrated with an apparent Mr of 10, 000 under reducing conditions. Amino acid sequencing revealed the N-terminal sequence EENIKKGKKXIRTPKI for HBGF-0.8-P1 and ENIKKGKKXIRT for HBGF-0.8-P2. These sequences corresponded, respectively, to residues 247-262 and 248-259 of the 349-residue predicted primary translation product of porcine connective tissue growth factor (pCTGF). 10-kDa CTGF-mediated fibroblast DNA synthesis was modulated by exogenous heparin, and CTGF-immunoreactive proteins of 10, 16, and 20 kDa were present in unfractionated uterine luminal flushings. These data reveal the identity of a novel growth factor in uterine fluids as a highly truncated form of CTGF and show that the N-terminal two-thirds of the CTGF primary translation product is not required for mitogenic activity or heparin binding.

Induction of anchorage independent growth by heparin-binding EGF-like growth factor (HB-EGF).

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is initially synthesized as a membrane bound protein that is subsequently processed to yield an approximately 74 amino acid secreted product. To investigate the biological activities of HB-EGF and its role(s) in tumor formation, the full-length HB-EGF cDNA was cloned under the regulation of the mouse metallothionein promoter and stably expressed in HB-EGF deficient mouse L cells. HB-EGF immunoreactive proteins of 21 and 24 kDa were observed from transfected MLC lysates, and these lysates exhibited the ability to bind to the EGF receptor, stimulate 3H-thymidine uptake in BALB/c-3T3 cells, and induce anchorage independent growth (AIG) of normal rat kidney (NRK) cells. Furthermore, NRK cells treated with either E. coli-derived or vaccinia virus-derived HB-EGF, as well as NRK cells directly transfected with the HB-EGF construct, demonstrated AIG. We conclude that HB-EGF is a potent growth factor capable of stimulating altered cell growth and anchorage independence.