The 5′-flanking sequence of the mouse epidermal growth factor gene has been isolated from a mouse genomic DNA library. S1 nuclease mapping indicated that the transcription start sites used in the submaxillary gland and the kidney are identical. Computer-aided sequence comparisons have indicated regions of the gene which may be involved in hormonal regulation.
Glucokinase (GK) plays a critical role in controlling blood glucose; GK activators have been shown to stimulate insulin secretion acutely both in vitro and in vivo. Sustained stimulation of insulin secretion could potentially lead to β-cell exhaustion; this study examines the effect of chronic GK activation on β-cells. Gene expression and insulin secretion were measured in rodent islets treated in vitro with GKA71 for 72 h. Key β-cell gene expression was measured in rat, mouse and global GK heterozygous knockout mouse islets (gk del/wt ). Insulin secretion, after chronic exposure to GKA71, was measured in perifused rat islets. GKA71 acutely increased insulin secretion in rat islets in a glucose-dependent manner. Chronic culture of mouse islets with GKA71 in 5 mmol/l glucose significantly increased the expression of insulin, IAPP, GLUT2, PDX1 and PC1 and decreased the expression of C/EBPβ compared with 5 mmol/l glucose alone. Similar increases were shown for insulin, GLUT2, IAPP and PC1 in chronically treated rat islets. Insulin mRNA was also increased in GKA71-treated gk del/wt islets. No changes in GK mRNA were observed. Glucose-stimulated insulin secretion was improved in perifused rat islets following chronic treatment with GKA71. This was associated with a greater insulin content and GK protein level. Chronic treatment of rodent islets with GKA71 showed an upregulation of key β-cell genes including insulin and an increase in insulin content and GK protein compared with glucose alone.
Aldo–keto reductases (AKRs) are multifunctional enzymes capable of acting on a wide variety of substrates, including sex steroids. AKRs having 20α-hydroxysteroid dehydrogenase (20α-HSD) activity can reduce progesterone to 20α-hydroxy-4-pregnen-3-one (20α-DHP), a metabolite with lower affinity for the progesterone receptor. The objective of this study was to investigate the regulation of equine AKR1C23 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The equine AKR1C23 cDNA was cloned and shown to encode a 322 amino acid protein that is conserved (71–81% identity) when compared with mammalian orthologs. RT-PCR/Southern blotting analyses were performed to study the regulation of AKR1C23 transcripts in equine preovulatory follicles isolated between 0 and 39 h after hCG treatment (ovulation occurring 39–42 h post-hCG). Results showed the presence of low AKR1C23 expression before hCG treatment, but a marked increase was observed in follicles obtained 12 h after hCG (P<0.05). Analyses of isolated preparations of granulosa and theca interna cells identified low mRNA expression in both cell types prior to hCG treatment, with granulosa cells clearly being the predominant site of follicular AKR1C23 mRNA induction. A specific polyclonal antibody was raised against a fragment of the equine protein and immunoblotting analyses showed an increase in AKR1C23 protein in granulosa cell extracts when comparing follicles isolated at 36 h post-hCG vs those collected prior to treatment, in keeping with mRNA results. Immunohistochemical data confirmed the induction of the enzyme in follicular cells after hCG treatment. The enzyme was tested for 20α-HSD activity and was shown to exhibit a K M of 3.12 μM, and a Vmax of 0.86 pmol/min per 10 μg protein towards progesterone. The levels of 20α-DHP measured in follicular fluid reflected this activity. Collectively, these results demonstrate for the first time that the gonadotropin-dependent induction of follicular luteinization is accompanied by an increase in AKR1C23 expression. Considering the 20α-HSD activity of AKR1C23, its regulated expression in luteinizing preovulatory follicles may provide a biochemical basis for the increase in ovarian 20α-DHP observed during gonadotropin-induced luteinization/ovulation. (The nucleotide sequence reported in this paper has been submitted to GenBank with accession number AY955082.)
A transgenic mouse line (EGF/Tag) has been established in which expression of SV40 T-antigen is directed by a 5·5 kb fragment of the 5′-flanking region of the mouse epidermal growth factor (EGF) gene. Of the two principal sites of EGF expression in mice, submaxillary gland and kidney, T-antigen mRNA and protein were detected in the former but not in the latter tissue of the EGF/Tag animals. T-antigen expression in the submaxillary gland was restricted to the EGF-producing cells of the granular convoluted tubules, and the oncoprotein induced hyperplasia of these cells. T-antigen levels were markedly higher in the submaxillary glands of male compared with female transgenic mice, suggesting that expression of the transgene was androgen-regulated, like the endogenous EGF gene. These results indicate that the 5·5 kb fragment upstream of the mouse EGF gene contains the DNA enhancer elements required for hormonally regulated expression in the submaxillary gland. Since the hyperplastic submaxillary glands of the EGF/Tag mice continue to synthesize EGF, these glands provide a tissue source from which it may prove possible to establish EGF-secreting cell lines for further in vitro studies of the mechanisms regulating expression of the EGF gene.
HL60 is a human promyeloid cell line capable of differentiating towards monocytes or granulocytes when treated with appropriate agents. Changes in insulin receptor number, affinity and mRNA levels were observed when HL60 cells were induced to differentiate with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or dimethylsulphoxide (DMSO). Total and high-affinity insulin receptor numbers decreased following treatment of HL60 cells with DMSO, whereas total insulin receptor number increased and high-affinity receptor number decreased in cells treated with TPA. Three distinct insulin receptor mRNA species of 9·1, 6·3 and 2·8 kb were identified in HL60 cells. The larger 9·1 and 6·3 kb species were increased in both TPA- and DMSO-treated HL60 cells, and the 2·8 kb mRNA was reduced in differentiated cells. Thus HL60 cells differentiated towards monocytes or granulocytes showed similar changes in the levels of individual insulin receptor mRNAs, but displayed contrasting alterations in low-affinity insulin binding. Three HL60 variant lines, which have different capacities to respond to inducers of monocyte and neutrophil differentiation, showed similar levels of total insulin receptors, but differed in their expression of high-affinity receptors. The data provide evidence for the existence of two distinct insulin receptors.
A portion of the pig epidermal growth factor (EGF) gene has been isolated and characterized. The nucleotide sequencies of exons 20 and 21, which encode the EGF region of the precursor protein, show 85% similarity with the human EGF gene sequence. In addition, conservation of the intron—exon boundaries between the two species was generally observed. Although the pig exon 21 appeared to lack a single nucleotide at its 5′ end relative to the human gene, sequences obtained by direct amplification of the genomic DNA around the 5′ end of this exon using the polymerase chain reaction, and from a pig EGF cDNA recombinant isolated from a kidney library, indicated that the deletion was probably a cloning artifact. Comparison of the predicted amino acid sequence of pig EGF with that of EGF from other species, as well as with several other polypeptides which bind to the EGF receptor, indicated conservation of Gly18, Tyr37, Gly39 and Arg41 in addition to all six cysteine residues and Leu47, which are known to be critical for biological activity. A synthetic gene encoding the predicted amino acid sequence of pig EGF was expressed in yeast. The recombinant polypeptide was shown to compete with 125I-labelled mouse EGF for binding to cells and to stimulate DNA synthesis in quiescent monolayers of Swiss 3T3 cells.
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