Gerhard Raab

Heparin-binding EGF-like growth factor.

HB-EGF is a heparin-binding member of the EGF family that was initially identified in the conditioned medium of human macrophages. Soluble mature HB-EGF is proteolytically processed from a larger membrane-anchored precursor and is a potent mitogen and chemotactic factor for fibroblasts, smooth muscle cells but not endothelial cells. HB-EGF activates two EGF receptor subtypes, HER1 and HER4 and binds to cell surface HSPG. The transmembrane form of HB-EGF is a juxtacrine growth and adhesion factor and is uniquely the receptor for diphtheria toxin. HB-EGF gene expression is highly regulated, for example by cytokines, growth factors, and transcription factors such as MyoD. HB-EGF has been implicated as a participant in a variety of normal physiological processes such as blastocyst implantation and wound healing, and in pathological processes such as tumor growth, SMC hyperplasia and atherosclerosis.

Heparin-binding EGF-like growth factor and ErbB signaling is essential for heart function

The heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of growth factors that binds to and activates the EGF receptor (EGFR) and the related receptor tyrosine kinase, ErbB4. HB-EGF-null mice (HBdel/del) were generated to examine the role of HB-EGF in vivo. More than half of the HBdel/del mice died in the first postnatal week. The survivors developed severe heart failure with grossly enlarged ventricular chambers. Echocardiographic examination showed that the ventricular chambers were dilated and that cardiac function was diminished. Moreover, HBdel/del mice developed grossly enlarged cardiac valves. The cardiac valve and the ventricular chamber phenotypes resembled those displayed by mice lacking EGFR, a receptor for HB-EGF, and by mice conditionally lacking ErbB2, respectively. HB-EGF–ErbB interactions in the heart were examined in vivo by administering HB-EGF to WT mice. HB-EGF induced tyrosine phosphorylation of ErbB2 and ErbB4, and to a lesser degree, of EGFR in cardiac myocytes. In addition, constitutive tyrosine phosphorylation of both ErbB2 and ErbB4 was significantly reduced in HBdel/del hearts. It was concluded that HB-EGF activation of receptor tyrosine kinases is essential for normal heart function.

Matrix Metalloproteinase-3 Releases Active Heparin-binding EGF-like Growth Factor by Cleavage at a Specific Juxtamembrane Site

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is synthesized as a membrane-anchored precursor that is cleaved to release the soluble mature growth factor. The two forms are active as juxtacrine and paracrine/autocrine growth factors, respectively. The enzymes that process the HB-EGF transmembrane form are unknown. Accordingly, an in vitro assay was established using a fusion protein in which alkaline phosphatase (AP) replaced the transmembrane and cytoplasmic domains of HB-EGF (HB-EGF JM-AP). The fusion protein was anchored to agarose beads coated with anti-AP antibodies. Several matrix metalloproteinases (MMPs) were tested for the ability to release soluble HB-EGF in the in vitrosystem. MMP-3 released soluble 12-kDa immunoreactive and mitogenic HB-EGF within 30 min. On the other hand neither MMP-2 nor MMP-9 had any cleavage activities. A non-cleavable mutant was prepared by replacing the juxtamembrane (JM) region of HB-EGF with the JM region of CD4. The mutant HB-EGF, which in its full-length form was as active a juxtacrine growth factor as was the wild type HB-EGF in vivo, was not cleaved by MMP-3 in the in vitro assay. The C-terminal portion of the cleaved HB-EGF JM-AP that remained attached to the anti-AP beads was N-terminally sequenced and the MMP-3 cleavage site was determined to be Glu151-Asn152, a site within the JM domain. MMP-3 treatment also released soluble HB-EGFin vivo from MC2 cells expressing transmembrane HB-EGF precursor, at a level of about 2-fold above control. It was concluded that MMP-3 cleaves HB-EGF at a specific site in the JM domain and that this enzyme might regulate the conversion of HB-EGF from being a juxtacrine to a paracrine/autocrine growth factor.

The Shedding of Membrane-anchored Heparin-binding Epidermal-like Growth Factor Is Regulated by the Raf/Mitogen-activated Protein Kinase Cascade and by Cell Adhesion and Spreading

Heparin-binding epidermal-like growth factor (HB-EGF) is synthesized as a transmembrane precursor (HB-EGFTM). The addition of phorbol ester (PMA, phorbol 12-myristate 13-acetate) to cells expressing HB-EGFTM results in the metalloproteinase-dependent release (shedding) of soluble HB-EGF. To analyze mechanisms that regulate HB-EGF shedding, a stable cell line was established expressing HB-EGFTM in which the ectodomain and the cytoplasmic tail were tagged with hemagglutinin (HA) and Myc epitopes, respectively (HB-EGFTMHA/Myc). HB-EGFTMHA/Myc cleavage was followed by the appearance of soluble HB-EGFHA in conditioned medium, the loss of biotinylated cell-surface HB-EGFTMHA/Myc, and the appearance of a Myc-tagged cytoplasmic tail fragment in cell lysates. By using this approach, several novel metalloproteinase-dependent regulators of HB-EGFTM shedding were identified as follows. (i) HB-EGFTMHA/Myc shedding induced by PMA was blocked by the mitogen-activated protein (MAP) kinase kinase inhibitor, PD98059. PMA activated MAP kinase within 5 min, but HB-EGFTMHA/Myc shedding did not occur until 20 min, suggesting that MAP kinase activation was a necessary step in the pathway of PMA-induced HB-EGFTM cleavage. (ii) Activation of an inducible Raf-1 kinase, ΔRaf-1:estrogen receptor, resulted in a rapid MAP kinase activation within 10 min and shedding of HB-EGFTMHA/Myc within 20–40 min. (iii) Serum induced MAP kinase activation and HB-EGFTMHA/Myc shedding that were inhibited by PD98059. (iv) Whereas PMA induced HB-EGFTMHA/Myc shedding in attached cells, no shedding occurred when the cells were placed in suspension. Shedding was fully restored shortly after cells were allowed to spread on fibronectin, and the extent of PMA-induced shedding increased with the extent of cell spreading. PMA induced the same level of MAP kinase activation whether the cells were attached or in suspension suggesting that although MAP kinase activation might be necessary for shedding, it was not sufficient. Taken together, these results suggest that there are two components of cell regulation that contribute to the shedding process, not previously recognized, the Raf-1/MAP kinase signal transduction pathway and cell adhesion and spreading.

Heparin-binding EGF-like growth factor is an autocrine growth factor for human urothelial cells and is synthesized by epithelial and smooth muscle cells in the human bladder.

The epidermal growth factor receptor (HER1) has been implicated in regenerative growth and proliferative diseases of the human bladder epithelium (urothelium), however a cognate HER1 ligand that can act as a growth factor for normal human urothelial cells (HUC) has not been identified. Here we show that heparin-binding EGF-like growth factor (HB-EGF), an activating HER1 ligand, is an autocrine regulator of HUC growth. This conclusion is based on demonstration of HB-EGF synthesis and secretion by primary culture HUC, identification of HER1 as an activatable HB-EGF receptor on HUC surfaces, stimulation of HUC clonal growth by HB-EGF, inhibition of HB-EGF-stimulated growth by heparin and of log-phase growth by CRM 197, a specific inhibitor of HB-EGF/HER1 interaction, and identification of human urothelium as a site of HB-EGF precursor (proHB-EGF) synthesis in vivo. ProHB-EGF expression was also detected in the vascular and detrusor smooth muscle of the human bladder. These data suggest a physiologic role for HB-EGF in the regulation of urothelial proliferation and regeneration subsequent to mucosal injury. Expression of proHB-EGF is also a feature of differentiated vascular and detrusor smooth muscle in the bladder. Because proHB-EGF is known to be the high affinity diphtheria toxin (DT) receptor in human cells, synthesis of the HB-EGF precursor by human urothelium also suggests the possibility of using the DT-binding sites of proHB-EGF as an in vivo target for the intraluminal treatment of urothelial diseases.

Extracellular calcium influx stimulates metalloproteinase cleavage and secretion of heparin‐binding EGF‐like growth factor independently of protein kinase C

The phorbol ester, tetradecanoyl‐phorbol 13‐acetate (TPA), stimulates rapid proteolytic processing of the transmembrane, pro‐ form of heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) at cell surfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB‐EGF secretion mechanism. To test this possibility, we expressed a chimeric protein, consisting of proHB‐EGF fused to placental alkaline phosphatase (AP) near the amino terminus of processed HB‐EGF, in NbMC‐2 prostate epithelial cells. The proHB‐EGF‐AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Secreted HB‐EGF‐AP bound to heparin and exhibited potent growth factor activity. The presence of the AP moiety allowed highly quantitative measurements of cleavage‐secretion responses of proHB‐EGF to extracellular stimuli. As expected, rapid secretion of HB‐EGF‐AP was induced in a time‐ and dose‐dependent manner by TPA. However, this was also observed with the Ca2+ionophore, ionomycin, suggesting the involvement of extracellular Ca2+ ions in the secretion mechanism. Ionomycin‐induced secretion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA‐mediated secretion effect was inhibited by staurosporine, GF109203X, and by pretreatment with TPA, but not by calcium chelation. A small secretion response was induced by thapsigargin, which releases Ca2+ from intracellular stores, but this was completely eliminated by extracellular calcium chelation. Ionomycin‐ and TPA‐induced HB‐EGF‐AP secretion was not dependent on the presence of the proHB‐EGF cytoplasmic domain and was specifically inhibited by the metalloproteinase inhibitors 1,10‐phenanthroline and tissue inhibitor of metalloproteinase‐1 (TIMP‐1). These data demonstrate that extracellular Ca2+ influx activates a membrane‐associated metalloproteinase to process proHB‐EGF by a pathway that does not require PKC. J. Cell. Biochem. 69:143–153, 1998.

Isolation of mouse stromal cells associated with a human tumor using differential diphtheria toxin sensitivity

Tumor vascularization is accompanied by the migration of stromal cells, including endothelial cells, smooth muscle cells, and fibroblasts, into the tumor. The biological contributions of stromal cells to tumor vascularization have not been well-defined, partly due to the difficulty of culturing stromal cells in the presence of large numbers of fast-growing tumor cells. To address this problem, a strategy was devised to kill tumor cells but not stromal cells. Advantage was taken of the observation that diphtheria toxin (DT) kills human but not rodent cells. Human melanoma (MMAN) tumor cells were injected subcutaneously into nude mice. The tumors were excised, homogenized, and treated with 50 ng/ml DT for 24 hours. Elimination of melanoma cells by DT treatment was demonstrated by lack of detectable levels of microphthalmia, a transcription factor that is a marker for melanoma cells. The murine stromal cells were viable and found to be mostly smooth muscle cells. These cells constituted about 1.5% of the MMAN tumor. RNase protection assays using a specific murine vascular endothelial growth factor probe confirmed the murine origin of the stromal cells. This method allows rapid isolation of stromal cells and should facilitate biochemical and genetic analysis of tumor-stromal interactions.