Integrins β₁, α₆, and α₃contribute to mechanical strain-induced differentiation of fetal lung type II epithelial cells via distinct mechanisms

Abstract

-Mechanical forces regulate lung maturation in the fetus by promoting type II epithelial differentiation. However, the cell surface receptors that transduce these mechanical cues into cellular responses remain largely unknown. When distal lung type II epithelial cells isolated from embryonic day 19 rat fetuses were cultured on flexible plates coated with laminin, fibronectin, vitronectin, collagen, or elastin and exposed to a level of mechanical strain (5%) similar to that observed in utero, transmembrane signaling responses were induced under all conditions, as measured by ERK activation. However, mechanical stress maximally increased expression of the type II cell differentiation marker surfactant protein C when cells were cultured on laminin substrates. Strain-induced alveolar epithelial differentiation was inhibited by interfering with cell binding to laminin using soluble laminin peptides (IKVIV or YIGSR) or blocking antibodies against integrin ␤1, ␣3, or ␣6. Additional studies were carried out with substrates coated directly with different nonactivating anti-integrin antibodies. Blocking integrin ␤ 1 and ␣6 binding sites inhibited both cell adhesion and differentiation, whereas inhibition of ␣ 3 prevented differentiation without altering cell attachment. These data demonstrate that various integrins contribute to mechanical control of type II lung epithelial cell differentiation on laminin substrates. However, they may act via distinct mechanisms, including some that are independent of their cell anchoring role. laminin; surfactant protein C LUNG GROWTH AND DEVELOPMENT during fetal life are critical for extrauterine survival. Premature infants are often born before sufficient lung maturation has occurred with resultant increased morbidity and mortality. Lung maturation, in part, is controlled mechanically. For example, fetal thoracic movements, which generate ϳ5% changes of the distal lung surface area (14, 15), and cell tractional forces exerted on extracellular matrix (ECM) within tissues have been shown to be critical regulators of fetal lung development (28,35,43).A key component of lung development is the differentiation of type II epithelial cells, the major source of pulmonary surfactant that prevents alveolar collapse during expiration. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and restoration of normal alveolar epithelium after lung injury (38). Past studies have shown that application of levels of mechanical strain that simulate the cell distortion produced by fetal breathing movements induces fetal type II epithelial cell maturation and that the ERK pathway mediates this response (41, 43). However, the mechanisms by which lung cells sense mechanical forces via mechanoreceptors to activate this intracellular differentiation pathway remain largely unknown.Interactions between pulmonary epithelial cells and ECM proteins such as laminin or fibronectin or different collagen subtypes modulate lung development (34). Laminin is a major glycoprotein component ...

Cigarette smoke causes lung vascular barrier dysfunction via oxidative stress-mediated inhibition of RhoA and focal adhesion kinase

“…The PAEC were propagated in MEM containing 10% FBS and sodium pyruvate. Alveolar type II epithelial cells were isolated from lungs of fetal C57BL/6 mice (embryonic days 17-18), as previously described (49). Briefly, after collagenase digestion, cell suspensions were sequentially filtered through 100-, 30-, and 15-m nylon meshes using screen cups (Sigma).…”

Strain-induced Differentiation of Fetal Type II Epithelial Cells Is Mediated via the Integrin α6β1-ADAM17/Tumor Necrosis Factor-α-converting Enzyme (TACE) Signaling Pathway

“…The presence of a vaginal plug was considered as day 0.5 of pregnancy. Type II cells were isolated as previously described (42). Briefly, after collagenase digestion, cell suspensions were sequentially filtered through 100-, 30-, and 15-m nylon meshes using screen cups (Sigma-Aldrich).…”

“…Numerous studies have confirmed that integrins play a central role in mechanotransduction in virtually all cell and tissue types (39 -41). Previous experiments from our laboratory have shown that specific integrin subtypes contribute to mechanical strain-induced differentiation of fetal type II epithelial cells (42).…”

DNA Microarray Reveals Novel Genes Induced by Mechanical Forces in Fetal Lung Type II Epithelial Cells

“…Fetal rat lungs were obtained from timed-pregnant Sprague-Dawley rats (Charles Rivers, Wilmington, MA) and E19 type II cells were isolated as previously described (6). Epithelial cells were then seeded onto Bioflex plates precoated with collagen-1 (Flexcell Corporation, Hillsborough, NC) and maintained in serum-free Dulbecco's modified Eagle's medium (DMEM) for 24 h. Plates containing adherent cells were mounted in a Flexercell FX-4000 Strain Unit (Flexcell Corp.).…”