The complex of Fas-associated factor 1 with Hsp70 stabilizes the adherens junction integrity by suppressing RhoA activation

Fas-associated factor 1 (FAF1) is a scaffolding protein that plays multiple functions, and dysregulation of FAF1 is associated withmany types of diseases such as cancers. FAF1 contains multiple ubiquitin-related domains (UBA, UBL1, UBL2, UAS, and UBX), eachdomain interacting with a specific partner. In particular, the interaction of UBL1 with heat shock protein 70 (Hsp70) is associatedwith tumor formation, although the molecular understanding remains unknown. In this study, the structural analysis revealed thatHis160 of FAF1 is important for its interaction with Hsp70. The association of Hsp70 with FAF1 is required for the interaction withIQGAP1. FAF1 negatively regulates RhoA activation by FAF1–Hsp70 complex formation, which then interacts with IQGAP1. Thesesteps play a key role in maintaining the stability of cell-to-cell junction. We conclude that FAF1 plays a critical role in the structureand function of adherens junction during tissue homeostasis and morphogenesis by suppressing RhoA activation, which induces theactivation of Rho-associated protein kinase, phosphorylation of myosin light chain, formation of actin stress fiber, and disruptionof adherens junction. In addition, depletion of FAF1 increased collective invasion in a 3D spheroid cell culture. These results provideinsightinto how the FAF1–Hsp70 complex acts as a novelregulator ofthe adherens junction integrity. The complex can be a potentialtherapeutic target to inhibit tumorigenesis and metastasis.

Soft Substrate Induces Endothelial Cell Inflammation and Disrupts Endothelium Integrity

Atherosclerosis (AS) is the main cause of death and disability all over the world. A lot of efforts have been devoted to treat AS, among which tissue engineering blood vessel materials, including artificial blood vessels, stents and vascular patches, have brought hope to ameliorate the symptoms in AS patients. However, there remains a large percentage of implantation failure due to the incompatibility of the material with the body. AS is a multi-factor related disease, and chronic inflammation is a major event that involves with its pathogenesis and development. Since previous studies suggested that the stiffness of the blood vessel might affect the inflammatory conditions, in this paper, we investigate the mechanism of how substrate stiffness could affect the inflammation response of the endothelial cells (ECs). Polyacrylamide (PA) based hydrogels at different concentrations were used as the culture substrate for ECs. The mRNA expression level of VCAM-1 and ICAM-1 was determined by qRT-PCR. EC chemotactic effect was evaluated by the number of THP-1 adhered to EC monolayer. The protein levels of IκBα and NF-κB were determined by western blotting analysis. The expression and localization of the major adherens junctions (AJs) proteins, VE-cadherin and β-catenin, were evaluated by western blotting and immunofluorescence staining. Our results showed that ECs cultured on soft substrate (1 kPa) demonstrated more chemotactic effect and the amount of the monocytes adhered to them was higher than that on harder substrate (20 kPa, p < 0.05). Moreover, NF-κB signaling pathway in ECs on 1 kPa substrate was more activated compared to those on 20 kPa substrate, with the IκBα protein expression level in the cytoplasm decreasing and NF-κB translocating more into the nuclear. In addition, the AJs of the endothelial monolayer changed with the substrate stiffness. Compared with ECs on normal substrate (20 kPa), the protein expression level of β-catenin decreased (p < 0.05), and immunofluorescence staining of VE-cadherin and β-catenin showed the AJs between the ECs on soft substrate (1 kPa) were punctuated. Taken together, our results suggested the stiffness of the substrate was important in regulating inflammation of the ECs and the integrity of the cell-cell junction. Therefore, the stiffness of the tissue engineering blood vessel material should be considered as an important criterium to avoid EC inflammation.

The septin complex links the catenin complex to the actin cytoskeleton for establishing epithelial cell polarity

Cell polarity is essential for spatially regulating of physiological processes in metazoans by which hormonal stimulation‒secretion coupling is precisely coupled for tissue homeostasis and organ communications.However,the molecular mechanisms underlying epithelial cell polarity establishment remain elusive.Here,we show that septin cytoskeleton interacts with catenin complex to organize a functional domain to separate apical from basal membranes in polarized epithelial cells.Using polarized epithelial cell monolayer as a model system with transepithelial electrical resistance as functional readout,our studies show that septins are essential for epithelial cell polarization.Our proteomic analyses discovered a novel septin‒catenin complex during epithelial cell polarization.The functional relevance of septin‒catenin complex was then examined in three-dimensional(3D)culture in which suppression of septins resulted in deformation of apical lumen in cysts,a hallmark seen in polarity-deficient 3D cultures and animals.Mechanistically,septin cytoskeleton stabilizes the association of adherens catenin complex with actin cytoskeleton,and depletion or disruption of septin cytoskeleton liberates adherens junction and polarity complexes into the cytoplasm.Together,these findings reveal a previously unrecognized role for septin cytoskeleton in the polarization of the apical‒basal axis and lumen formation in polarized epithelial cells.