Epithelial 3D-spheroids as a tool to study air pollutant-induced lung pathology.

Cigarette smoke (CS) and air pollutants (AP) activate pathological processes in bronchial epithelial cells resulting in lung function decline which severely impacts human health. Knowledge about the molecular mechanism(s) by which CS and AP induce pathology is limited. Our previous studies in 2D cultures of human bronchial epithelial (BEAS-2B) cells showed that CS exposure activates transforming growth factor-ß1 (TGF-ß1) release and signaling. Furthermore, CS exposure reduced the expression of E-cadherin, which was prevented by applying a TGF-ß1 neutralizing antibody. Exposure of BEAS-2B cells cultured in 2D to diesel exhaust particles (DEP) increased TGF-ß1 protein expression and reduced the expression of epithelial cell markers, whereas mesenchymal markers are upregulated. Conventional 2D cell culture may, however, not fully reflect the physiology of bronchial epithelial cells in vivo. To simulate the in vivo situation more closely we cultured the bronchial epithelial cells in a 3D environment in the current study. Treatment of epithelial spheroids with TGF-ß resulted in reduced E-cadherin and increased collagen I expression, indicating the activation of epithelial-to-mesenchymal transition (EMT). Similarly, exposure of spheroids to DEP induced and EMT-like phenotype. Collectively, our data indicate AP induces an EMT-like phenotype of BEAS-2B cells in 3D spheroid cultures. This opens new avenues for drug development for the treatment of lung diseases induced by AP. The 3D spheroid cell culture is a novel, innovative and physiologically relevant model for culturing a variety of cells. It is a versatile tool for both high-throughput studies and for identifying molecular mechanisms involved in bronchial epithelial cell (patho)physiology.

A-Kinase Anchoring Proteins Diminish TGF-ß1/Cigarette Smoke-Induced Epithelial-To-Mesenchymal Transition.

Epithelial-to-mesenchymal transition (EMT) plays a role in chronic obstructive pulmonary diseases (COPD). Cyclic adenosine monophosphate (cAMP) can inhibit transforming growth factor-ß1 (TGF-ß1) mediated EMT. Although compartmentalization via A-kinase anchoring proteins (AKAPs) is central to cAMP signaling, functional studies regarding their therapeutic value in the lung EMT process are lacking. The human bronchial epithelial cell line (BEAS-2B) and primary human airway epithelial (pHAE) cells were exposed to TGF-ß1. Epithelial (E-cadherin, ZO-1) and mesenchymal markers (collagen Ӏ, α-SMA, fibronectin) were analyzed (mRNA, protein). ELISA measured TGF-ß1 release. TGF-ß1-sensitive AKAPs Ezrin, AKAP95 and Yotiao were silenced while using siRNA. Cell migration was analyzed by wound healing assay, xCELLigence, Incucyte. Prior to TGF-ß1, dibutyryl-cAMP (dbcAMP), fenoterol, rolipram, cilostamide, and forskolin were used to elevate intracellular cAMP. TGF-ß1 induced morphological changes, decreased E-cadherin, but increased collagen Ӏ and cell migration, a process that was reversed by the inhibitor of δ/epsilon casein kinase I, PF-670462. TGF-ß1 altered (mRNA, protein) expression of Ezrin, AKAP95, and Yotiao. St-Ht31, the AKAP antagonist, decreased E-cadherin (mRNA, protein), but counteracted TGF-ß1-induced collagen Ӏ upregulation. Cigarette smoke (CS) increased TGF-ß1 release, activated TGF signaling, augmented cell migration, and reduced E-cadherin expression, a process that was blocked by TGF-ß1 neutralizing antibody. The silencing of Ezrin, AKAP95, and Yotiao diminished TGF-ß1-induced collagen Ӏ expression, as well as TGF-ß1-induced cell migration. Fenoterol, rolipram, and cilostamide, in AKAP silenced cells, pointed to distinct cAMP compartments. We conclude that Ezrin, AKAP95, and Yotiao promote TGF-ß1-mediated EMT, linked to a TGF-ß1 release by CS. AKAP members might define the ability of fenoterol, rolipram, and cilostamide to modulate the EMT process, and they might represent potential relevant targets in the treatment of COPD.