The soluble and membrane-anchored forms of heparin-binding epidermal growth factor-like growth factor appear to play opposing roles in the survival and apoptosis of human luteinized granulosa cells

Abstract

This study aims to investigate the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and its role in regulating apoptosis of human luteinized granulosa cells (LGC). By using RT-PCR and immunocytochemistry, the expression of HB-EGF and the EGF receptor family was demonstrated. HER4, one of the two cognate receptors for HB-EGF, was found translocated into the nucleus. HB-EGF exists in two forms, the precursor membrane-anchored form and the mature secreted form. Addition of recombinant HB-EGF, which acts as the secreted form, into the cell culture inhibited apoptosis and appeared to stimulate mitosis, indicating that the secreted form is potentially an anti-apoptotic factor and a mitogen for LGC. In contrast, CRM197, a specific inhibitor for the interaction between HB-EGF and the EGF receptor, inhibited rather than enhanced apoptosis, suggesting that the membrane-anchored form constitutively functions as a pro-apoptotic factor for LGC. Furthermore, the finding that apoptosis inhibition by CRM197 in the aggregate cells was much more pronounced than in the single cells indicates that pro-apoptotic activity was carried out in a juxtacrine fashion, as would be expected for the membrane-anchored form of HB-EGF. These data suggest that HB-EGF may be a unique regulator of LGC apoptosis, with two functionally opposing products arising from the same gene.

Changes in the expression and cytological localization of betacellulin and its receptors (ErbB-1 and ErbB-4) in the trophoblasts in human placenta over the course of pregnancy

“…The soluble mature form is regarded as an autocrine or a paracrine growth factor in various tissues, whereas the transmembrane form is thought to have juxtacrine activity, which is recognized as the interactions of an immobilized growth factor with neighboring cells (40). Indeed, recent investigation regarding HB-EGF has shown that the biological effects of the transmembrane form on human luteinized granulosa cells are different from those of the soluble form (41). Similar to other members of the EGF family, BTC is synthesized as a transmembrane precursor, which is subject to proteolytic cleavage of its ectodomain to produce a soluble mature growth factor (13).…”

The heparin-binding domain of HB-EGF mediates localization to sites of cell-cell contact and prevents HB-EGF proteolytic release

“…Interestingly, mutation of the HBD of HB-EGF not only reversed the growth inhibition of HB-EGF transfection, but also led to cell proliferation, with a 42% increase in [ 3 H]thymidine incorporation. Because HB-EGF juxtacrine signaling has been reported to be growth inhibitory (Iwamoto et al, 1999;Pan et al, 2002), we hypothesize that mutation of the HBD decreases the juxtacrine interaction by decreasing the concentration of pro-HB-EGF at sites of cell-cell contact. Additionally, because mutation of the HBD increased the rate of ligand cleavage from the cell surface, we hypothesize that an increase in autocrine signaling stimulates cell proliferation.…”

“…HB-EGF juxtacrine signaling and autocrine and/or paracrine signaling have been shown to elicit different phenotypes, with autocrine and/or paracrine activity leading to cell proliferation and migration (Higashiyama et al, 1991;Yahata et al, 2006), and juxtacrine activity causing growth inhibition and in some cases apoptosis (Iwamoto et al, 1999;Pan et al, 2002). HB-EGF proteolytic cleavage and subsequent autocrine signaling drives physiologically beneficial migration of a variety of tissue cells, such as keratinocytes (Shirakata et al, 2005;Tokumaru et al, 2000), corneal epithelial cells (Block et al, 2004;Xu et al, 2004), smooth-muscle cells (Bakken et al, 2009), peritoneal mesothelial cells (Faull et al, 2001) and mesenchymal stem cells (Ozaki et al, 2007) during wound healing.…”

Thioredoxin, an antioxidant redox protein, in ovarian follicles of women undergoing <i>in vitro</i> fertilization

“…GAPDH was chosen as the internal standard to control for mRNA loading and to normalize TRX. The oligonucleotide sequences used for the forward and reverse primers, melting temperature (Tm in °C), PCR cycle number, and predicted product size (bp) were as follows [14,15]: TRX: forward, 5'-TGAAGCAGATCGAGAGCAAGAC-3', reverse, 5'-TTCATTAATGGRGGCRRCAAGC-3', 60°C, 30 cycles, 305 bp; cTRXR: forward, 5'-GAAGATCTTCCCAAGTCCTATGAC-3', reverse, 5'-ATTTGTTGCCTTAATCCTGTGAGG-3', 64°C, 27 cycles, 420 bp; and GAPDH: forward, 5'-TCACCATCTTCCAGGAGCG-3', reverse, 5'-CTGCTTCACCACCTTCTTGA-3', 60°C, 30 cycles, 572 bp. For each unknown sample, the concentration of TRX, TRXR, and GAPDH was determined from the corresponding standard curves.…”