Abstract
The enzyme serine palmitoyltransferase (SPT; EC 2.3.1.50), which catalyzes the first committed and rate-limiting step in sphingolipid synthesis, is up-regulated in the epidermis as part of the homeostatic repair in response to permeability barrier perturbation. Moreover, UVB exposure, which also perturbs the barrier, up-regulates sphingolipid synthesis, but the basis for this increase is not known. The recent isolation of cDNAs for SPT (i.e., LCB1 and LCB2) allow molecular regulation studies to be performed. Therefore, we determined whether UVB exposure alters mRNA, protein, or activity levels for SPT in cultured human keratinocytes (CHKs) as a mechanism for regulation of epidermal sphingolipid synthesis. In CHK, transcripts for both LCB1 (3.0 kb) and LCB2 (2.3 kb) are evident by Northern blot analysis, and UVB exposure (23 mJ/cm2) induces a delayed 1.8 to 3.3-fold increase in LCB2 mRNA levels (P < 0.01) 48 h after treatment versus non-irradiated control cells. In contrast, neither LCB1 nor a second LCB2 transcript (8.0 kb) changed significantly. Likewise, Lcb2 protein levels (by Western blot analysis), as well as SPT activity, increase in parallel with the increased LCB2 mRNA. Finally, incorporation of [14C]-acetate into sphingolipids was increased significantly 48 h after UVB treatment. Together, these results demonstrate that CHKs respond to UVB by increasing sphingolipid synthesis, primarily through increases in both LCB2 mRNA and protein levels, leading to increased SPT activity. These results demonstrate one mechanism (UVB) whereby SPT is regulated at the molecular level, and suggest further that epidermis up-regulates sphingolipid synthesis at both the mRNA and protein levels in response to UVB.—Farrell, A. M., Y. Uchida, M. M. Nagiec, I. R. Harris, R. C. Dickson, P. M. Elias, and W. M. Holleran. UVB irradiation up-regulates serine palmitoyltransferase in cultured human keratinocytes. J. Lipid Res. 1998. 39: 2031–2038.
Highlights
The enzyme serine palmitoyltransferase (SPT; EC 2.3.1.50), which catalyzes the first committed and ratelimiting step in sphingolipid synthesis, is up-regulated in the epidermis as part of the homeostatic repair in response to permeability barrier perturbation
We first determined the baseline expression of LCB1 and LCB2 mRNAs in non-UVB-treated cultured human keratinocytes (CHKs) in order to compare the size of expressed transcripts with those described previously
Recovery from the UVB insult is paralleled by an increase in both epidermal sphingolipid synthesis and epidermal SPT activity in vivo [24]
Summary
The enzyme serine palmitoyltransferase (SPT; EC 2.3.1.50), which catalyzes the first committed and ratelimiting step in sphingolipid synthesis, is up-regulated in the epidermis as part of the homeostatic repair in response to permeability barrier perturbation. We determined whether UVB exposure alters mRNA, protein, or activity levels for SPT in cultured human keratinocytes (CHKs) as a mechanism for regulation of epidermal sphingolipid synthesis. Serine palmitoyltransferase (SPT) (EC 2.3.1.50), the enzyme that catalyzes the initial committed step in de novo sphingolipid synthesis, is highly active both in murine epidermis and in cultured human keratinocytes [2, 3]. Both SPT activity and sphingolipid synthesis increase during the process of barrier recovery after acute damage [4], and the increase in sphingolipid synthesis is required for permeability barrier homeostasis [5]. Whereas UV irradiation appears to up-regulate epidermal sphingolipid production, the mechanism(s) by which UVB mediates these process(es) have not been addressed
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