Keratinocyte-specific Expression of Fatty Acid Transport Protein 4 Rescues the Wrinkle-free Phenotype in Slc27a4/Fatp4 Mutant Mice

Meei-Hua Lin,Nicholas O Davidson,Casey L Moulson,J Michael White,Elizabeth P Newberry,Jeffrey H Miner

doi:10.1074/jbc.m701779200

Meei-Hua Lin, Nicholas O Davidson + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m701779200

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Abstract

FATP4 (fatty acid transport protein 4; also known as SLC27A4) is the most widely expressed member of a family of six long chain fatty acid transporters. FATP4 is highly expressed in enterocytes and has therefore been proposed to be a major importer of dietary fatty acids. Two independent mutations in Fatp4 cause mice to be born with thick, tight, shiny, "wrinkle-free" skin and a defective skin barrier; they die within hours of birth from dehydration and restricted movements. In contrast, induced keratinocyte-specific deficiency of FATP4 in adult mice causes only mild skin abnormalities. Therefore, whether the loss of FATP4 from skin or a systemic gestational metabolic defect causes the severe skin defects and neonatal lethality remain important unanswered questions. To investigate the basis for the phenotype, we first generated wild-type tetraploid/mutant diploid aggregates that should lead to rescue of any abnormalities caused by loss of FATP4 from the placenta. However, the skin phenotype was not ameliorated. We then generated transgenic mice expressing exogenous FATP4 either widely or specifically in suprabasal keratinocytes, and we bred the transgenes onto the Fatp4(-/-) background. Both modes of FATP4 expression led to rescue of the neonatally lethal skin defects, and the resulting mice were viable and fertile. Keratinocyte expression of an FATP4 variant with mutations in the acyl-CoA synthetase domain did not provide any degree of rescue. We conclude that expression of FATP4 with an intact acyl-CoA synthetase domain in suprabasal keratinocytes is necessary for normal skin development and that FATP4 functions in establishing the cornified envelope.

Highlights

We found that FATP4 expressed in suprabasal keratinocytes, under the control of the involucrin promoter, is sufficient to rescue the lethality and most of the skin defects of Fatp4Ϫ/Ϫ mice
Expression of Fatp4 is most prominent in trophoblasts (Fig. 1G), the cells that transport all substances between mother and fetus
Tetraploid embryos were derived from matings between wild-type mice, and these were aggregated with diploid embryos derived from matings between Fatp4ϩ/Ϫ mice, of which 1⁄4 are expected to be Fatp4Ϫ/Ϫ embryos

Summary

EXPERIMENTAL PROCEDURES

Tetraploid Aggregations—Tetraploid aggregations were performed as described [13]. Briefly, wild-type ICR two-celled embryos were electrofused to form tetraploids and cultured for 24 h. The Ivl-⌬Fatp transgene was produced from the Fatp cDNA with the S247G and T249G mutations introduced using primers containing the mutations and a QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Sections were mounted in 90% glycerol containing 0.1ϫ PBS and 1 mg/ml p-phenylenediamine. Sections were deparaffinized in XS-3 xylene substitute (StatLab Medical Product, Lewisville, TX), rehydrated in graded ethanols, fixed again in 4% paraformaldehyde, and digested in 5 ␮g/ml proteinase K in PBS for 10 min. Sections were washed in MABT, in NTMT (10 mM NaCl, 50 mM MgCl2, 100 mM Tris, pH 9.5, 0.1% Tween 20), in NTMT with 2 mM levamisol, and color-developed in BM Purple (Roche Applied Science) with 2 mM levamisol and 0.1% Tween 20 for 1 h to 3 days in the dark. Lipids were extracted with chloroform and methanol [20] and characterized by electrospray ionization mass spectrometry as described [21, 22]

RESULTS

Hair counts from rescued mutant and control mice

DISCUSSION