TY - JOUR
T1 - Modulation of phospholipase D-mediated phosphatidylglycerol formation by differentiating agents in primary mouse epidermal keratinocytes
AU - Zheng, Xiangjian
AU - Ray, Sagarika
AU - Bollag, Wendy B.
N1 - Funding Information:
We thank Dr. Nancy Pryer for her generous gift of PLD-2-overexpressing insect cell membranes. We also gratefully acknowledge Dr. Maurice Pechet for providing the 1,25-dihydroxyvitamin D 3 . This work was supported by National Institutes of Health (National Institute of Arthritis, Musculoskeletal and Skin Diseases) grant #AR 45212 (to WBB) and was performed in partial fulfillment of the requirements for the degree of Doctor in Philosophy in the Molecular Medicine Graduate Program (XZ). This work has been presented in part at the 60th and 62nd annual meetings of the Society for Investigative Dermatology, May 1999 in Chicago, IL and May 2001 in Washington, DC.
PY - 2003/12/7
Y1 - 2003/12/7
N2 - The major component of the epidermis, keratinocytes, must continuously proliferate and differentiate to form the mechanical and water permeability barrier of the skin. Our previous data have suggested a potential role in these processes for phospholipase D (PLD), an enzyme that hydrolyzes phospholipids to generate phosphatidic acid. In the presence of primary alcohols, PLD also catalyzes a transphosphatidylation reaction to produce phosphatidylalcohols, and this characteristic has been exploited to monitor the activity of PLD in intact cells. In this report, PLD was demonstrated to utilize the physiological alcohol glycerol to form phosphatidylglycerol (PG) in vitro. In intact primary murine epidermal keratinocytes treated for 24 h with elevated extracellular Ca2+ levels, but not 1,25-dihydroxyvitamin D3, incubation with radioactive glycerol resulted in an increase in PLD-mediated radiolabeled PG production. This effect was dose-dependent and biphasic, with maximal PG formation detected after exposure to an intermediate (125 μM) Ca 2+ concentration. Furthermore, the biphasic nature of the response was due, in part, to a corresponding biphasic change in glycerol uptake. Finally, short-term treatment of keratinocytes with phorbol 12-myristate 13-acetate (PMA) failed to increase PG synthesis and inhibited glycerol uptake. Since (1) PMA is reported to activate PLD-1 to a greater extent than PLD-2, (2) 1,25-dihydroxyvitamin D3 increases the expression/activity of PLD-1 in keratinocytes, and (3) PLD-2 is co-localized with a glycerol channel in keratinocyte membrane microdomains, we speculate that radiolabeled PG production from radioactive glycerol is a measure of PLD-2 activation in these cells. Our results also suggest that PLD-mediated PG synthesis may be regulated at the level of both PLD activity and alcohol substrate availability via changes in glycerol uptake.
AB - The major component of the epidermis, keratinocytes, must continuously proliferate and differentiate to form the mechanical and water permeability barrier of the skin. Our previous data have suggested a potential role in these processes for phospholipase D (PLD), an enzyme that hydrolyzes phospholipids to generate phosphatidic acid. In the presence of primary alcohols, PLD also catalyzes a transphosphatidylation reaction to produce phosphatidylalcohols, and this characteristic has been exploited to monitor the activity of PLD in intact cells. In this report, PLD was demonstrated to utilize the physiological alcohol glycerol to form phosphatidylglycerol (PG) in vitro. In intact primary murine epidermal keratinocytes treated for 24 h with elevated extracellular Ca2+ levels, but not 1,25-dihydroxyvitamin D3, incubation with radioactive glycerol resulted in an increase in PLD-mediated radiolabeled PG production. This effect was dose-dependent and biphasic, with maximal PG formation detected after exposure to an intermediate (125 μM) Ca 2+ concentration. Furthermore, the biphasic nature of the response was due, in part, to a corresponding biphasic change in glycerol uptake. Finally, short-term treatment of keratinocytes with phorbol 12-myristate 13-acetate (PMA) failed to increase PG synthesis and inhibited glycerol uptake. Since (1) PMA is reported to activate PLD-1 to a greater extent than PLD-2, (2) 1,25-dihydroxyvitamin D3 increases the expression/activity of PLD-1 in keratinocytes, and (3) PLD-2 is co-localized with a glycerol channel in keratinocyte membrane microdomains, we speculate that radiolabeled PG production from radioactive glycerol is a measure of PLD-2 activation in these cells. Our results also suggest that PLD-mediated PG synthesis may be regulated at the level of both PLD activity and alcohol substrate availability via changes in glycerol uptake.
KW - Aquaporin-3
KW - Glycerol
KW - Phorbol 12-myristate 13-acetate (PMA)
KW - Phospholipase D-2 (PLD-2)
KW - Transphosphatidylation
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U2 - 10.1016/j.bbamcr.2003.08.006
DO - 10.1016/j.bbamcr.2003.08.006
M3 - Article
C2 - 14654225
AN - SCOPUS:0344255818
SN - 0167-4889
VL - 1643
SP - 25
EP - 36
JO - Biochimica et Biophysica Acta - Molecular Cell Research
JF - Biochimica et Biophysica Acta - Molecular Cell Research
IS - 1-3
ER -