Human Epidermal Growth Factor Receptor-1 Expression Renders Chinese Hamster Ovary Cells Sensitive to Alternative Aldosterone Signaling

Krug, Alexander W.; Schuster, Claudia; Gaßner, Birgit; Freudinger, Ruth; Mildenberger, Sigrid; Troppmair, Jakob; Gekle, Michael

doi:10.1074/jbc.m208851200

Cited by 79 publications

(91 citation statements)

References 37 publications

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“…In this study, we employed a heterologous expression system to over express human HER3 and HER3/HER2 chimeric receptors in CHO cells to examine the role of intracellular domains of HER3 in heterodimerization and HER3 downstream signaling. CHO cells are known to not express endogenous EGFR (Krug et al, 2002). This is confirmed in the CHO cells used in our study by RT-PCR.…”

Section: Discussionsupporting

confidence: 74%

HER3 intracellular domains play a crucial role in HER3/HER2 dimerization and activation of downstream signaling pathways

et al. 2012

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Dimerization among the EGFR family of tyrosine kinase receptors leads to allosteric activation of the kinase domains of the partners. Unlike other members in the family, the kinase domain of HER3 lacks key amino acid residues for catalytic activity. As a result, HER3 is suggested to serve as an allosteric activator of other EGFR family members which include EGFR, HER2 and HER4. To study the role of intracellular domains in HER3 dimerization and activation of downstream signaling pathways, we constructed HER3/HER2 chimeric receptors by replacing the HER3 kinase domain (HER3-2-3) or both the kinase domain and the C-terminal tail (HER3-2-2) with the HER2 counterparts and expressed the chimeric receptors in Chinese hamster ovary (CHO) cells. While over expression of the intact human HER3 transformed CHO cells with oncogenic properties such as AKT/ERK activation and increased proliferation and migration, CHO cells expressing the HER3-2-3 chimeric receptor showed significantly reduced HER3/HER2 dimerization and decreased phosphorylation of both AKT and ERK1/2 in the presence of neuregulin-1 (NRG-1). In contrast, CHO cells expressing the HER3-2-2 chimeric receptor resulted in a total loss of downstream AKT activation in response to NRG-1, but maintained partial activation of ERK1/2. The results demonstrate that the intracellular domains play a crucial role in HER3's function as an allosteric activator and its role in downstream signaling.

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Section: Discussionsupporting

confidence: 74%

HER3 intracellular domains play a crucial role in HER3/HER2 dimerization and activation of downstream signaling pathways

et al. 2012

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“…Subsequently, the relative number of cells in each well was determined using crystal violet staining of the nuclei, allowing normalization of ERK1/2 phosphorylation for cell number. 23 Briefly, the cells were washed twice with PBS/Triton and twice with PBS. After drying the wells for 5 min, 100 ml of crystal violet solution was added for 30 min at room temperature.…”

Section: Western Blot Analysismentioning

confidence: 99%

“…23,24 Briefly, samples were matched for protein content, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a nitrocellulose membrane. Subsequently, membranes were immunostained with rabbit anti-phospho-ERK1/2 antibody (1:1000; Cell Signalling, Danvers, MA, USA), anti-ERK1/2 antibody (1:1,000; Cell Signalling) or anti-StAR antibody (1:1000; Santa Cruz Technology, Santa Cruz, CA, USA).…”

Section: Western Blot Analysismentioning

confidence: 99%

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Human adipocytes induce an ERK1/2 MAP kinases-mediated upregulation of steroidogenic acute regulatory protein (StAR) and an angiotensin II — sensitization in human adrenocortical cells

et al. 2007

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Objectives: Hypertension is a major complication of overweight with frequently elevated aldosterone levels in obese patients. Our previous work suggests a direct stimulation of adrenal aldosterone secretion by adipocytes. Owing to aldosterone's important role in maintaining blood pressure homeostasis, its regulation in obesity is of major importance. One objective was to determine the signaling mechanisms involved in adipocyte-induced aldosterone secretion. In addition to a direct stimulation, a sensitization toward angiotensin II (AngII) might be involved. The second objective was to determine a possible adipokinesinduced sensitization of human adrenocortical cells to AngII. Design: Human subcutaneous adipocytes and adrenocortical cells, and the adrenocortical cell line NCI-H295R were used. Adrenocortical cells were screened for signal transduction protein expression and phosphorylation. Subsequently, steroidogenic acute regulatory protein (StAR), cAMP response element-binding protein (CREB), cAMP and phosphorylated extracellular regulated kinase were analyzed by Western blot, enzyme-linked immunosorbent assay, quantitative PCR, reporter gene assay and confocal microscopy to investigate their role in adipocyte-mediated aldosterone secretion. Results: AngII-mediated aldosterone secretion was largely increased by preincubating H295R cells with adipocyte secretory products. StAR mRNA and StAR protein were upregulated in a time-dependent way. This steroidogenic effect was independent of the cAMP-protein kinase A (PKA) pathway as cellular cAMP was unaltered and inhibition of PKA by H89 failed to reduce aldosterone secretion. However, CREB reporter gene activity was moderately elevated. Upregulation of StAR was accompanied by ERK1/2 MAP kinase activation and nuclear translocation of the kinases. Inhibition of MAP kinase by UO126 abolished adipokine-stimulated aldosterone secretion from primary human adrenocortical and H295R cells, and inhibited StAR gene activity. Adipokines stimulated steroidogenesis also in primary human adrenocortical cells, supporting a role in human physiology and/or pathology. Conclusions: Adipokines induce aldosterone secretion from human adrenocortical cells and sensitization of the cells to stimulation by AngII, possibly mediated via ERK1/2-dependent upregulation of StAR activity. This stimulation of aldosterone secretion could be one link between overweight and inappropriately elevated aldosterone levels.

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“…Over-expression of Heparin-binding EGF is detected in ARPKD and EGF antagonistic antibodies suppress the mitogenic activity of cleared cystic fluid [35]. The abundance of EGFR in cells from the renal tubule is responsive to aldosteronedependent transcription [36] and expression of EGFR is necessary to permit the stimulation of rapid signalling responses by aldosterone in CHO cells [37]. The 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 7 activation of ERK1/2 signalling by aldosterone in cells derived from the distal nephron has been described by several groups including our own [38,39] and this response has been linked to trans-activation of EGFR [40].…”

Section: Aldosterone Egfr and Polycystic Kidney Diseasementioning

confidence: 99%

Aldosterone as a renal growth factor

2010

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Human Epidermal Growth Factor Receptor-1 Expression Renders Chinese Hamster Ovary Cells Sensitive to Alternative Aldosterone Signaling

Abstract: The epidermal growth factor (EGF) regulates cell proliferation, differentiation, and ion transport using ERK1/2 as a downstream effector. Furthermore, the EGF receptor (

Cited by 79 publications

References 37 publications

HER3 intracellular domains play a crucial role in HER3/HER2 dimerization and activation of downstream signaling pathways

HER3 intracellular domains play a crucial role in HER3/HER2 dimerization and activation of downstream signaling pathways

Human adipocytes induce an ERK1/2 MAP kinases-mediated upregulation of steroidogenic acute regulatory protein (StAR) and an angiotensin II — sensitization in human adrenocortical cells

Aldosterone as a renal growth factor

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