Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli

The expression of the recombinant diphtheria toxin mutant CRM197 in bacteria other than Corynebacterium diphtheriae has proven to be difficult. Here we propose a new and alternative procedure for the production of full-length CRM197 in Escherichia coli. The present study relates specifically to the expression of an artificial sequence and to a method for the isolation and purification of the corresponding protein. In particular, a synthetic gene coding for CRM197, bearing a short histidine tag and optimized for E. coli codon usage, was cloned in the pET9a vector. Accordingly, the over-expression of the protein was simply induced with arabinose in E. coli BL21AI. The recombinant protein was insoluble and always found inside protein aggregates, which were solubilised using urea. Surprisingly, the expression of CRM197, devoid of the short tag, always failed. Following a refolding step, the his-tagged CRM197 was purified by affinity and gel-filtration chromatography and the purity of the final preparation reached 95%. Interestingly, the recombinant protein features DNase activity, indicating that the presence of the tag is not affecting its biochemical properties. However, the removal of the synthetic tag could be easily obtained by incubating the target protein with a proper quantity of a commercial enterokinase.     

Activation of Pyk2 by CaM kinase II in cultured hypothalamic neurons and gonadotroph cells

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic GnRH neurons and stimulates a GnRH receptor in gonadotroph cells and GnRH neurons. The GnRH receptor belongs to the G-protein-coupled receptors, and stimulation of the GnRH receptor activates extracellular signal-regulated protein kinase (ERK). We reported previously that the δ2 isoform of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase IIδ2) was involved in GnRH-induced ERK activation in cultured GnRH neurons (GT1–7 cells). Recently, we found that GnRH treatment of GT1–7 cells activated proline-rich tyrosine kinase 2 (Pyk2), and Pyk2 was involved in ERK activation. In the current study, we examined the possibility that CaM kinase IIδ2 might activate Pyk2. Knockdown of CaM kinase IIδ2 and KN93, an inhibitor of CaM kinases, inhibited the GnRH-induced activation of Pyk2. In the case of cultured gonadotroph cells (αT3-1 cells), knockdown of CaM kinase IIβ’e inhibited GnRH-induced Pyk2 activation. In addition, our inhibitor studies indicated that Pyk2 and CaM kinase II were involved in the GnRH-induced shedding of proHB-EGF in GT1–7 cells. These results suggested that CaM kinase II activated the ERK pathway through Pyk2 activation and HB-EGF production in response to GnRH.     

Short form of the heparin-binding EGF-like growth factor: Communication II

The structure of the green monkey Chlorocebus aethiops heparin-binding EGF-like growth factor (HB-EGF) gene was compared with that of the corresponding human gene. Exon 3a, characteristic of the short form of HB-EGF (SF-HB-EGF), was mapped between exons 3 and 4, approximately 700 bp away from the latter. In several human and simian cell lines, most of the SF-HB-EGF mRNA proved to lack exons 4 and 5, specific to the HB-EGF mRNA. In contrast to the HB-EGF mRNA, the SF-HB-EGF mRNA occurred predominantly in the P, rather than L, form, which codes for a protein with a different propeptide structure. Labeled SF-HB-EGF competed with HB-EGF and EGF for binding to the surface of A431 cells, suggesting its interaction with the specific EGF receptor. The results indicate that SF-HB-EGF plays a specific role in cell signaling.     

Heparin-binding EGF-like growth factor: a juxtacrine growth factor

Heparin-binding EGF-like growth factor (HB-EGF), which belongs to the EGF-family growth factors, is synthesized as a membrane-anchored form (proHB-EGF). Proteolytic cleavage of proHB-EGF at the extracellular domain yields the soluble form of HB-EGF (sHB-EGF). ProHB-EGF is not only the precursor molecule for sHB-EGF but also a biologically active molecule itself. Recent studies indicate that proHB-EGF has unique properties distinct from the soluble form. ProHB-EGF forms a complex with membrane proteins including a tetramembrane spanning protein: CD9, an adhesion molecule integrin: 3β1, and heparan sulfate proteoglycans. The complex is localized at the cell–cell contact site, suggesting that proHB-EGF may function in cell-to-cell signaling by a juxtacrine mechanism. In an in vitro model system, proHB-EGF showed growth inhibitory activity, while sHB-EGF was growth stimulatory. Ectodomain shedding, conversion of the membrane-anchored form into the soluble form, is regulated by multiple signaling pathways. All these characteristics imply that proHB-EGF and sHB-EGF are used in different ways. In vivo functions of sHB-EGF and proHB-EGF have been largely undefined, but recent studies implicate them in a variety of physiological processes including blastocyst implantation and wound healing.     

Gastrin mediates resistance to hypoxia-induced cell death in xenografts of the human colorectal cancer cell line LoVo

Aim: Gastrins act as growth factors for the normal and neoplastic colorectal mucosa. The aim of this study was to determine the role of gastrins in the response of human colorectal cancer (CRC) cells to hypoxia in vitro and in vivo. Methods: Expression of the gastrin gene in the human CRC cell line LoVo was examined under normoxia and hypoxia by quantitative PCR and by radioimmunoassay. Gastrin expression was knocked down with shRNA, and the effect on cell proliferation was measured by cell counting, on cell apoptosis by annexin V staining, and on cell migration by Boyden chamber assay. The effect of gastrin knockdown on tumourigenesis in mouse xenografts was analysed by measurement of tumour volumes and weights, and by immunohistochemistry. Results: Gastrin gene expression in LoVo cells was stimulated by hypoxia via binding of hypoxia-inducible factor-1α to the gastrin promoter. The viability of gastrin knockdown cells exposed to hypoxia (1% O₂) in vitro was diminished because of loss of resistance against hypoxia-induced apoptosis, and the effect was partly reversed by treatment with non-amidated, but not amidated, gastrin. Conditioned medium from control LoVo cells under hypoxia simulated proliferation but not migration, and the effect was blocked by an inhibitor of non-amidated gastrins, but not by an inhibitor of amidated gastrins. In xenografts in mice exposed to hypoxia (10% O₂) for 21 days, tumour necrosis was significantly increased by knocking down gastrin expression. Conclusion: These results provide evidence that non-amidated gastrins are involved in the adaptation of CRCs to hypoxic microenvironments through increasing resistance to apoptosis.     

HB-EGF: a unique mediator of embryo-uterine interactions during implantation

An implantation-competent blastocyst, several hours prior to its attachment on the uterine wall, transmits signals to surrounding uterine cells and vice-versa to initiate a two-way interaction. The language of this precocious dialogue is versatile, taking advantage of secreted molecules for long-range interactions and membrane-bound molecules for more immediate interactions. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified as an early messenger of implantation which uses both modes of communication. In this review, we discuss the footprint of HB-EGF as to how it was initially identified as a mediator of implantation and how it initiates embryo-uterine interactions during this process.     

AngiotensinII mediates cardiomyocyte hypertrophic growth pathways via MMP-dependent HB-EGF liberation

Pathological cardiac stimulation by angiotensinII (AngII) can cause left ventricular hypertrophy, a major independent risk factor for heart attack and death. We have previously reported that AngII exerts its hypertrophic effects by usurping the epidermal growth factor (EGF) signalling pathway via metalloprotease-dependent transactivation. However, the EGF-like ligand responsible for AngII-mediated transactivation and cardiac hypertrophy remains to be identified. Using phosphorylated ERK1/2 as a read-out of growth pathway activation and an alkaline phosphatase-tagged Heparin-Binding EGF-like Growth Factor (HB-EGF) reporter construct to examine AngII-mediated liberation, we provide evidence that HB-EGF is the soluble growth factor involved in AngII-induced left ventricular hypertrophy.