Mechanical activation of lung epithelial cells through the ion channel Piezo1 activates the metalloproteinases ADAM10 and ADAM17 and promotes growth factor and adhesion molecule release.

In the lung, pulmonary epithelial cells undergo mechanical stretching during ventilation. The associated cellular mechanoresponse is still poorly understood at the molecular level. Here, we demonstrate that activation of the mechanosensitive cation channel Piezo1 in a human epithelial cell line (H441) and in primary human lung epithelial cells induces the proteolytic activity of the metalloproteinases ADAM10 and ADAM17 at the plasma membrane. These ADAMs are known to convert cell surface expressed proteins into soluble and thereby play major roles in proliferation, barrier regulation and inflammation. We observed that chemical activation of Piezo1 promotes cleavage of substrates that are specific for either ADAM10 or ADAM17. Activation of Piezo1 also induced the synthesis and ADAM10/17-dependent release of the growth factor amphiregulin (AREG). In addition, junctional adhesion molecule A (JAM-A) was shed in an ADAM10/17-dependent manner resulting in a reduction of cell contacts. Stretching experiments combined with Piezo1 knockdown further demonstrated that mechanical activation promotes shedding via Piezo1. Most importantly, high pressure ventilation of murine lungs increased AREG and JAM-A release into the alveolar space, which was reduced by a Piezo1 inhibitor. Our study provides a novel link between stretch-induced Piezo1 activation and the activation of ADAM10 and ADAM17 in lung epithelium. This may help to understand acute respiratory distress syndrome (ARDS) which is induced by ventilation stress and goes along with perturbed epithelial permeability and release of growth factors.

Bioprinting-associated pulsatile hydrostatic pressure elicits a mild proinflammatory response in epi- and endothelial cells.

During nozzle-based bioprinting, like inkjet and microextrusion, cells are subjected to hydrostatic pressure for up to several minutes. The modality of the bioprinting-related hydrostatic pressure is either constant or pulsatile depending on the technique. We hypothesized that the difference in the modality of hydrostatic pressure affects the biological response of the processed cells differently. To test this, we used a custom-made setup to apply either controlled constant or pulsatile hydrostatic pressure on endothelial and epithelial cells. Neither bioprinting procedure visibly altered the distribution of selected cytoskeletal filaments, cell-substrate adhesions, and cell-cell contacts in either cell type. In addition, pulsatile hydrostatic pressure led to an immediate increase of intracellular ATP in both cell types. However, the bioprinting-associated hydrostatic pressure triggered a pro-inflammatory response in only the endothelial cells, with an increase of interleukin 8 (IL-8) and a decrease of thrombomodulin (THBD) transcripts. These findings demonstrate that the settings adopted during nozzle-based bioprinting cause hydrostatic pressure that can trigger a pro-inflammatory response in different barrier-forming cell types. This response is cell-type and pressure-modality dependent. The immediate interaction of the printed cells with native tissue and the immune system in vivo might potentially trigger a cascade of events. Our findings, therefore, are of major relevance in particular for novel intra-operative, multicellular bioprinting approaches.

Cortical tension regulates desmosomal morphogenesis.

Mechanical stability is a fundamental and essential property of epithelial cell sheets. It is in large part determined by cell-cell adhesion sites that are tightly integrated by the cortical cytoskeleton. An intimate crosstalk between the adherens junction-associated contractile actomyosin system and the desmosome-anchored keratin intermediate filament system is decisive for dynamic regulation of epithelial mechanics. A major question in the field is whether and in which way mechanical stress affects junctional plasticity. This is especially true for the desmosome-keratin scaffold whose role in force-sensing is virtually unknown. To examine this question, we inactivated the actomyosin system in human keratinocytes (HaCaT) and canine kidney cells (MDCK) and monitored changes in desmosomal protein turnover. Partial inhibition of myosin II by para-nitro-blebbistatin led to a decrease of the cells' elastic modulus and to reduced desmosomal protein turnover in regions where nascent desmosomes are formed and, to a lower degree, in regions where larger, more mature desmosomes are present. Interestingly, desmosomal proteins are affected differently: a significant decrease in turnover was observed for the desmosomal plaque protein desmoplakin I (DspI), which links keratin filaments to the desmosomal core, and the transmembrane cadherin desmoglein 2 (Dsg2). On the other hand, the turnover of another type of desmosomal cadherin, desmocollin 2 (Dsc2), was not significantly altered under the tested conditions. Similarly, the turnover of the adherens junction-associated E-cadherin was not affected by the low doses of para-nitro-blebbistatin. Inhibition of actin polymerization by low dose latrunculin B treatment and of ROCK-driven actomyosin contractility by Y-27632 treatment also induced a significant decrease in desmosomal DspI turnover. Taken together, we conclude that changes in the cortical force balance affect desmosome formation and growth. Furthermore, they differentially modulate desmosomal protein turnover resulting in changes of desmosome composition. We take the observations as evidence for a hitherto unknown desmosomal mechanosensing and mechanoresponse pathway responding to an altered force balance.

Guiding cell adhesion and motility by modulating cross-linking and topographic properties of microgel arrays.

Biomaterial-driven modulation of cell adhesion and migration is a challenging aspect of tissue engineering. Here, we investigated the impact of surface-bound microgel arrays with variable geometry and adjustable cross-linking properties on cell adhesion and migration. We show that cell migration is inversely correlated with microgel array spacing, whereas directionality increases as array spacing increases. Focal adhesion dynamics is also modulated by microgel topography resulting in less dynamic focal adhesions on surface-bound microgels. Microgels also modulate the motility and adhesion of Sertoli cells used as a model for cell migration and adhesion. Both focal adhesion dynamics and speed are reduced on microgels. Interestingly, Gas2L1, a component of the cytoskeleton that mediates the interaction between microtubules and microfilaments, is dispensable for the regulation of cell adhesion and migration on microgels. Finally, increasing microgel cross-linking causes a clear reduction of focal adhesion turnover in Sertoli cells. These findings not only show that spacing and rigidity of surface-grafted microgels arrays can be effectively used to modulate cell adhesion and motility of diverse cellular systems, but they also form the basis for future developments in the fields of medicine and tissue engineering.