No abstract

Keratinocyte growth factor (KGF, FGF-7) is a potent mitogen for epithelial cells. We instilled recombinant human KGF to determine the effects of KGF on alveolar epithelial cells. Left lungs of adult rats were instilled intrabronchially with KGF (5 mg/kg) or normal saline. KGF instillation resulted in epithelial cell hyperplasia, and the alveolar bromodeoxyuridine (BrdU) labeling index peaked at 35% on day 2 after instillation. The mRNA levels for the surfactant proteins (SPs) SP-A, SP-B, and SP-D were increased in whole lung tissue on days 1 and 2 after KGF treatment and then returned to control levels on days 3-7. SP-C mRNA levels were increased on days 2-5 after KGF instillation. However, all surfactant protein mRNAs were reduced in type II cells isolated from rats instilled with KGF 2 or 3 days before isolation. These observations were confirmed by in situ hybridization. Instillation of KGF also increased the amount of SP-A and SP-D in lavage fluid. Transcripts for CC10, the 10-kDa Clara cell protein, were decreased. KGF increases the mRNA for the surfactant proteins per lung because of type II cell hyperplasia, but the mRNA per cell is slightly diminished as measured in isolated cells or estimated by in situ hybridization.

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 phosphatidylcholin, 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 α (C/EBPα) and C/EBPδ as well as SREBP-1c (ADD-1), but not PPARγ. These changes in C/EBPα and C/EBPδ 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/EBPα, C/EBPδ, 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.

We have investigated whether the loss of differentiated function observed in adult rat alveolar type II cells cultured on a substratum that promotes cell spreading and flattening represents a reversible phenotypic change. Cells were cultured for 4 and 8 days in association with fetal rat lung fibroblast feeder layers on either attached collagen gels, which promote the loss of differentiated function, or on floating collagen gels, which support differentiation. A fifth group of cultures were maintained as attached gels for 4 days, then detached and cultured as floating gels for the remaining 4 days. Expression of mRNAs for surfactant proteins A, B, and C, patterns of phospholipid biosynthesis, rates and patterns of protein synthesis, and cell morphology were evaluated as markers of differentiation. Without exception, detaching the gels after 4 days in culture resulted in significant recovery of differentiated characteristics, demonstrating that type II cells modulate differentiated function in response to the culture environment. The results are discussed in relation to the importance of normal cell architecture to normal cell function and to the possible in vitro progression of type II cells to type I cells.

Secretion of surfactant proteins A and D (SP-A and SP-D) has been difficult to study in vitro because a culture system for maintaining surfactant secretion has been difficult to establish. We evaluated several growth factors, corticosteroids, rat serum, and a fibroblast feeder layer for the ability to produce and maintain a polarized epithelium of type II cells that secretes SP-A and SP-D into the apical medium. Type II cells were plated on a filter insert coated with an extracellular matrix and were cultured at an air-liquid interface. Keratinocyte growth factor (KGF) stimulated type II cell proliferation and secretion of SP-A and SP-D more than fibroblast growth factor-10 (FGF-10), hepatocyte growth factor (HGF), or heparin-binding epidermal-like growth factor (HB-EGF). Cells cultured in the presence of KGF and rat serum with or without fibroblasts had high surfactant protein mRNA levels and exhibited a high level of SP-A and SP-D secretion. Dexamethasone inhibited type II cell proliferation but increased expression of SP-B. In the presence of KGF, rat serum, and dexamethasone, the mRNAs for the surfactant proteins were maintained at high levels. Secretion of SP-A and SP-D was found to be independent of phospholipid secretion.