No abstract

The hypoxia-inducible factor (HIF) is a key player in a transcriptional pathway that controls the hypoxic response of mammalian cells. Post-translational modification of the ␣ subunit of HIF determines its half-life and activity. Among the multiple reported modifications, acetylation, by an acetyltransferase termed arrest-defective-1 protein (ARD1), has been reported to decrease HIF-1␣ stability and therefore impact on hypoxic gene expression. In contrast, we report that both overexpression and silencing of ARD1 had no impact on the stability of HIF-1␣ or -2␣ and that cells silenced for ARD1 maintained hypoxic nuclear localization of HIF-1␣. In addition, we show that the ARD1 mRNA and protein levels are not regulated by hypoxia in several human tumor cell lines, including cervical adenocarcinoma HeLa cells, fibrosarcoma HT1080 cells, adenovirustransformed human kidney HEK293 cells, and human breast cancer MCF-7 cells. Using two model systems ((a) wild-type and HIF-1␣-null mouse embryo fibroblasts and (b) HeLa cells silenced for HIF-1␣ or -2␣ by RNA interference), we demonstrate that the level of expression of the ARD1 protein is independent of HIF-1␣ and -2␣. We also demonstrate that ARD1 is a stable, predominantly cytoplasmic protein expressed in a broad range of tissues, tumor cell lines, and endothelial cells. Taken together, our findings demonstrate that ARD1 has limited, if any, impact on the HIF signaling pathway.

Cellular fibronectin (FN) variants harboring one or two alternatively spliced Extra Domains (B and A) play a central bioregulatory role during development, repair processes and fibrosis. Yet, how the Extra Domains impact fibrillar assembly and function of the molecule remains unclear. Leveraging a unique biological toolset and image analysis pipeline for direct comparison of the variants, we demonstrate that the presence of one or both Extra Domains impacts FN assembly, function and physical properties of the matrix. When presented to FN-null fibroblasts, Extra Domain-containing variants differentially regulate pH homeostasis, survival, and TGF-β signaling by tuning the magnitude of cellular responses, rather than triggering independent molecular switches. Numerical analyses of fiber topologies highlight significant differences in variant-specific structural features and provide a first step for the development of a generative model of FN networks to unravel assembly mechanisms and investigate the physical and functional versatility of extracellular matrix landscapes.