Related Experiment Videos
Inhibition of protein tyrosine phosphatase activity blocks shape change & steroidogenesis in Y1 cells
B J Whitehouse1, S L Gyles, C J Burns
1Endocrinology & Reproduction Research Group, School of Biomedical Sciences, King's College London.
Abstract:
Y1 adrenocortical cells respond to forskolin stimulation with increases in steroid secretion and change of shape. The rapid rounding of flat, adherent cells which occurs is known to involve dephosphorylation of the focal adhesion protein, paxillin. We have investigated the effects of a tyrosine phosphatase inhibitor, calpeptin (CP) on steroidogenesis and shape change in Y1 cells. Forskolin treatment (FSK, 2 microM) caused marked rounding of Y1 cells (FSK = 76.3 +/- 1.5% cells rounded after 30 minutes, untreated = 2.9 +/- 0.7 % rounded); calpeptin pretreatment (CP; 100 ug/ml) had little effect on shape (9.6 +/- 2.4% rounded) but blocked the rounding response to FSK (32.1 +/- 2.1% rounded. Calpetin also eliminated the steroidogenic response to FSK ( FSK = 242 +/- 14% control ; FSK + CP = 113 +/- 18% control ) without affecting production of steroid from membrane permeant 22R-OH-cholesterol. The results support the view that dephosphorylation of paxillin is important in the rounding response and provide evidence for the involvement of tyrosine-phosphatase activity in cyclic AMP-stimulated steroidogenesis in Y1 cells.
Insights
Forskolin stimulates steroid secretion and cell rounding in Y1 cells, involving paxillin dephosphorylation. A tyrosine phosphatase inhibitor, calpeptin, blocks these responses, indicating phosphatase involvement in cyclic AMP-stimulated steroidogenesis.
Area of Science:
- Cell Biology
- Endocrinology
- Biochemistry
Background:
- Y1 adrenocortical cells exhibit shape changes and increased steroid secretion upon forskolin stimulation.
- This forskolin-induced cell rounding is linked to the dephosphorylation of paxillin, a focal adhesion protein.
Purpose of the Study:
- To investigate the role of tyrosine phosphatase activity in forskolin-induced steroidogenesis and shape change in Y1 cells.
- To determine the effect of the tyrosine phosphatase inhibitor calpeptin on these cellular responses.
Main Methods:
- Y1 cells were treated with forskolin (FSK) and/or calpeptin (CP).
- Cell shape changes (rounding) and steroid secretion were quantified.
- Steroid production from a membrane-permeant precursor was assessed to control for general steroidogenic capacity.
Main Results:
- Forskolin significantly increased cell rounding and steroid secretion.
- Calpeptin pretreatment partially blocked forskolin-induced cell rounding.
- Calpeptin abolished the forskolin-stimulated steroidogenic response without affecting basal steroid production.
Conclusions:
- Tyrosine phosphatase activity is implicated in the cyclic AMP-mediated steroidogenesis in Y1 cells.
- Paxillin dephosphorylation is crucial for the cell rounding response to forskolin.
- Inhibition of tyrosine phosphatases disrupts steroidogenesis in response to forskolin stimulation.