Keratinocyte growth factor and the transcription factors C/EBP alpha, C/EBP delta, and SREBP-1c regulate fatty acid synthesis in alveolar type II cells

Abstract

Strategies to stimulate endogenous surfactant production require a detailed understanding of the regulation of lipogenesis in alveolar type II cells. We developed culture conditions in which keratinocyte growth factor (KGF) stimulates fatty acid and phospholipid synthesis. KGF stimulated acetate incorporation into phosphatidylcholine, disaturated phosphatidylcholine, and phosphatidylglycerol more than 5% rat serum alone. To determine the mRNA levels of lipogenic enzymes and transport proteins, we analyzed gene expression by oligonucleotide microarrays. KGF increased the mRNA levels for fatty acid synthase, stearoyl-CoA desaturase-1 (SCD-1), and epidermal fatty acid-binding protein more than rat serum alone. In addition, KGF increased the mRNA levels of the transcription factors CCAAT/enhancer-binding protein alpha (C/EBPalpha) and C/EBPdelta as well as SREBP-1c (ADD-1), but not PPARgamma. These changes in C/EBPalpha and C/EBPdelta were confirmed by in situ hybridization. SCD-1 was also found to be highly expressed in alveolar type II cells in vivo. Furthermore, KGF increased protein levels of fatty acid synthase, C/EBPalpha, C/EBPdelta, SREBP-1, epidermal fatty acid-binding protein, and SCD. Finally, the liver X receptor agonist T0901317 increased acetate incorporation and SREBP-1 but not SREBP-2 protein levels. In summary, KGF stimulates lipogenesis in type II cells by a coordinated expression of lipogenic enzymes and transport proteins regulated by C/EBP isoforms and SREBP-1c.

Figure 1

SREBP-1c mRNA level is increased in the differentiated cultures. Type II cells were cultured in 5% RS with and without 10 ng/ml KGF and 10^–8 M Dex in the apical-access system. The mRNA levels were determined by an RPA as described in Methods. The values were normalized to 18S RNA and then compared with the value for 5% RS, which was given a level of 1. (a) A representative RPA. Lanes 1–4 show samples from type II cells cultured in the apical-access system and harvested on day 7. Lane 5 shows a sample from freshly isolated type II cells. (b) The summary of three independent experiments normalized to 5% RS. *P < 0.05 after adjustment for repeated-measures ANOVA.

Figure 2

C/EBPα, SCD-1, and SCD-2 mRNA are increased in type II cells in response to KGF. (a–l) Type II cells were cultured in 5% RS with or without KGF for 6 days in the apical-access system. The monolayers were fixed with 4% paraformaldehyde, processed for in situ hybridization with sense and antisense probes, and stained with hematoxylin. In a (bright field) and c (dark field), the results for RS alone are shown. The expression is very low but higher than with the sense probe (data not shown). In b (bright field) and d (dark field), there is increased expression of C/EBPα in the presence of RS plus KGF. Similarly, KGF stimulates the expression of SCD-1 (e–h) and SCD-2 (i–l). (m and n) The expression of SCD-1 is shown in the normal lung in bright field (m) and dark field (n). Specific cells in the alveolar region are heavily labeled, and the distribution of these cells is the same as that of SP-C, a marker restricted to type II cells. The micrographs were taken at ×400.

Figure 3

C/EBPα, C/EBPδ, and SREBP-1 protein levels are increased in the differentiated cultures. Type II cells were cultured in 1% CS-FBS alone (lane 1), plus KGF (lane 2), plus 5% RS (lane 3), or plus 5% RS and KGF (lane 4) for 6 days in the apical-access system under air/liquid conditions. The cells were extracted and the protein expression measured by Western analysis as described in Methods. Because matrix proteins within the gel precluded a reliable measurement of cellular protein, loading of individual lanes was based on the amount of actin. A representative example of at least three determinations is shown.

Figure 4

Fatty acid synthesis is stimulated by the LXR agonist T0901317. Type II cells were plated on Matrigel in 5% RS and then cultured in 1% CS-FBS with or without KGF and with or without varying doses of T0901317 and appropriate vehicle controls. (a) T0901317 increased acetate incorporation in a dose-dependent manner in 1% CS-FBS and at the highest dose in the presence of KGF. The results are normalized to the value of 1% CS-FBS and are means ± SEM for five independent experiments. *P < 0.05 vs. the appropriate DMSO control without the LXR agonist. (b) Protein expression is shown by immunoblotting. The conditions are the same as in a. Lanes 1, 2, 7, and 8 contain DMSO without T0901317 as shown in a. Protein loading is normalized to the amount of actin. A representative sample of five independent experiments is shown.