Expression and regulation of phospholipase D in the human keratinocyte cell line HaCaT

Abstract

The generation of lipid second messengers via phosphatidylcholine (PC)-specific phospholipase D (PLD) has emerged as an important step leading to transduction of extracellular signals. In the present investigation the expression of human cytosolic PLD isoenzymes in the immortalized human keratinocyte cell line HaCat was determined. At the mRNA level we found the expression of hPLD1b and for the first time in human cells also the expression of hPLD2. For further analysis of enzyme expression at the protein level, hPLD1 peptide fragments were synthesized and specific antibodies were generated (rabbit) to be used for detection of hPLD1 in Western blot experiments. Furthermore, small G-proteins were found to be involved in the regulation of PLD activity in HaCaT cells using the guanine nucleotide analogue GTPQ QS.z 1998 Federation of European Biochemical Societies.

Rat pancreatic acinar cells express a cytosolic phospholipase D1b isoform that is not regulated by cholecystokinin

“…At present, information on the expression levels, cellular localization and function of PLD1 and PLD2 proteins is limited. Using a specific antibody raised against N-and C-terminal sequences of human PLD1 [28] the present study showed that both rat pancreatic acinar cells and CHO cells contain substantial amounts of this PLD isoform. RT-PCR on total RNA suggested that, in both cases, the 1b splice variant of PLD1 was present.…”

“…Proteins were transferred overnight to polyvinylidene difluoride membranes (Immobillon P, Millipore, Bedford, Mass., USA). The blots were incubated overnight with rabbit anti-hPLD antibody [28]. The anti-912 body was kindly provided by Prof. Dr. C.C.…”

Preparation of Primary Cultures of Mouse Epidermal Keratinocytes and the Measurement of Phospholipase D Activity

“…Both PLD1 and PLD2 are expressed in keratinocytes (42,43), and a correlation has been uncovered between sustained PLD activation and the induction of keratinocyte differentiation (44). PLD1 is highly conserved across species (92 % identity, 97 % similarity) and is upregulated in response to 1,25-dihydroxyvitamin D 3 (42).…”

Mechanism of vitamin D3‐induced transcription of phospholipase D1 in HaCat human keratinocytes

“…The PLD signaling has been thought to mediate late differentiation events in epidermal keratinocytes. In HaCaT human keratinocyte cells, both the expression of PLD1 and PLD2 mRNA and the presence of PLD1 protein were reported briefly [7], but the sequential change in PLD isozymes during VitD 3 -induced differentiation and the regulatory mechanism of PLD1 gene expression have not been investigated.…”