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The Hippo pathway controls cell-cell interaction and organ size by regulating cell proliferation. The study aimed to determine whether Hippo/YAP and NF-kappa B/RELA signaling interact. Here, we have demonstrated that native YAP1/TEAD and RELA proteins biochemically and functionally interact with each other in human LNCaP and C4-2 cell lines. Our co-immunoprecipitation (co-IP), western blot (WB), and proximity ligation assay (PLA) showed that endogenous YAP/TEAD and RELA physically interact within the cell. Our immunofluorescence assays revealed that the expression of YAP1 and RELA proteins overlapped in the cytoplasm and the nucleus. Combined treatment of cells with RANKL (receptor activator of nuclear factor-kappa Β ligand) and androgen hormone enhanced YAP1 and RELA colocalization and interaction, as demonstrated by co-IP/WB experiments. Moreover, our PLA confirmed that co-treatment of cells with androgen and SDF1a (stromal cell-derived factor 1 alpha) or RANKL increased YAP1 and RELA interaction cytoplasm and nucleus compared with controls. Our promoter-reporter assays showed that the knockdown of YAP1 by siRNA significantly reduced the activity of an NF-Kappa B responsive promoter-reporter gene. We also showed that controlled expression of MST1/STK4, a potent inhibitor of YAP1, attenuated the NF-Kappa B promoter reporter activity. Our unbiased bioinformatics analysis of the chromatin immunoprecipitation data has revealed that YAP/TEAD and NF-kappa B signaling regulates several genes. These findings suggest that interaction between the Hippo/YAP and NF-Kappa B/RELA plays a critical role in broad cellular biology.
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Engineered endosymbionts that alter mammalian cell surface marker, cytokine and chemokine expression
Abstract Developing modular tools that direct mammalian cell function and activity through controlled delivery of essential regulators would improve methods of guiding tissue regeneration, enhancing cellular-based therapeutics and modulating immune responses. To address this challenge, Bacillus subtilis was developed as a chassis organism for engineered endosymbionts (EES) that escape phagosome destruction, reside in the cytoplasm of mammalian cells, and secrete proteins that are transported to the nucleus to impact host cell response and function. Two synthetic operons encoding either the mammalian transcription factors Stat-1 and Klf6 or Klf4 and Gata-3 were recombined into the genome of B. subtilis expressing listeriolysin O (LLO) from Listeria monocytogenes and expressed from regulated promoters. Controlled expression of the mammalian proteins from B. subtilis LLO in the cytoplasm of J774A.1 macrophage/monocyte cells altered surface marker, cytokine and chemokine expression. Modulation of host cell fates displayed some expected patterns towards anti- or pro-inflammatory phenotypes by each of the distinct transcription factor pairs with further demonstration of complex regulation caused by a combination of the EES interaction and transcription factors. Expressing mammalian transcription factors from engineered intracellular B. subtilis as engineered endosymbionts comprises a new tool for directing host cell gene expression for therapeutic and research purposes.
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- Award ID(s):
- 2200991
- PAR ID:
- 10436584
- Date Published:
- Journal Name:
- Communications Biology
- Volume:
- 5
- Issue:
- 1
- ISSN:
- 2399-3642
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1038/s42003-022-03851-6
