Integration of Growth Factor, Extracellular Matrix, and Retinoid Signals during Bronchial Epithelial Cell Differentiation

Epithelial cell differentiation is a complex process that results from the integration of growth factor-, hormone-, and extracellular matrix (ECM)-derived signals (for reviews see references 17 and 38). The respiratory epithelium provides a good model system for the study of signal integration, since normal bronchial epithelial cell differentiation requires the correct combination of growth factors, retinoids, and ECM (73, 74). In vivo, the upper airways are populated with basal, ciliated, and mucosecretory (goblet) cells (reviewed in reference 24), which rest on a thin basement membrane and a thick lamina propria rich in ECM molecules such as collagens, laminin, fibronectin, and heparan sulfate proteoglycans. Vitamin A (retinol)-deficient animals develop lesions along their upper airways in which mucociliary cells are replaced with highly keratinized squamous epithelia (44, 72), indicating a critical role for retinoids in normal respiratory cell differentiation. When grown ex vivo on plastic dishes in defined serum-free media without retinoids, cultures of normal tracheal/bronchial epithelial cells undergo squamous metaplasia upon reaching confluence (30, 54). Although retinoid treatment inhibits expression of the squamous phenotype under these culture conditions (28, 30, 54), it does not stimulate mucociliary differentiation. For mucosecretory cell differentiation ex vivo, tracheal/bronchial epithelial cells must be cultured on type I collagen gels in the presence of retinoids (29, 55, 73) but with limited amounts of growth factors, since high doses of epidermal growth factor (EGF), for example, are inhibitory (23, 74). Growth factors signal by binding to and increasing the catalytic activity of receptor tyrosine kinases (RTKs) (for a review, see reference 34). Autophosphorylation of the RTK on its cytoplasmic tail results in the recruitment of several secondary signaling molecules. These usually include the SHC-GRB2-SOS and/or GRB2-SOS complexes, which participate in Ras activation, phosphatidylinositol 3′ kinase, SHP-2, and phospholipase C-γ. Ultimately, several downstream events occur, including activation of the mitogen-activated protein kinases (MAPKs) Erk1 and Erk2. ECM transmits signals via various cell adhesion molecules (for a review, see reference 57). Integrins are the most widely studied of this group of molecules (for a review, see reference 10). Much is known about the signaling events that are evoked immediately upon integrin ligation and attachment of cells to ECM. Typically, acute engagement of integrins leads to the activation of protein tyrosine kinases (PTKs) such as FAK and Src family PTKs, the subsequent recruitment of many of the same signaling molecules that are recruited to RTKs, and the activation of downstream pathways such as phosphatidylinositol 3′ kinase (35) and MAPK (9, 59). In contrast, little is known about the specific effects of long-term cell-ECM interactions on signaling pathways. Retinoids signal by binding to two classes of receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which belong to the nuclear hormone receptor superfamily of transcription factors (for a review, see reference 8). At least three genes encode each type of receptor (denoted α, β, γ), and typical responses are transduced through binding of RAR-RXR heterodimers to retinoic acid response elements (RAREs) in the promoters of target genes. Although much is known about how growth factors, retinoids, and ECM signal in isolation, virtually nothing is understood about how these disparate signals are integrated to direct complex responses such as differentiation and/or how signal integration is perturbed under pathological conditions such as cancer. We investigated the molecular basis for signal integration during mucosecretory differentiation of normal human bronchial epithelial cells (NHBEs). We found striking differences in the established paradigms for growth factor, retinoid, and ECM signaling pathways, which largely have been elucidated in studies using immortalized and/or transformed cell lines. These studies stress the importance of using normal primary cell systems to study general epithelial signal transduction pathways and provide new insight into how divergent pathways may cooperate to regulate normal physiologic responses such as differentiation in vivo.