Inhibition of Retinoic Acid Signaling in Proximal Tubular Epithelial cells Protects against Acute Kidney Injury by Enhancing Kim-1-dependent Efferocytosis.

Retinoic acid receptor (RAR) signaling is essential for mammalian kidney development, but in the adult kidney is restricted to occasional collecting duct epithelial cells. We now show there is widespread reactivation of RAR signaling in proximal tubular epithelial cells (PTECs) in human sepsis-associated acute kidney injury (AKI), and in mouse models of AKI. Genetic inhibition of RAR signaling in PTECs protects against experimental AKI but is associated with increased expression of the PTEC injury marker, Kim-1. However, Kim-1 is also expressed by de-differentiated, proliferating PTECs, and protects against injury by increasing apoptotic cell clearance, or efferocytosis. We show that the protective effect of inhibiting PTEC RAR signaling is mediated by increased Kim-1 dependent efferocytosis, and that this is associated with de-differentiation, proliferation, and metabolic reprogramming of PTECs. These data demonstrate a novel functional role that reactivation of RAR signaling plays in regulating PTEC differentiation and function in human and experimental AKI.

Regulation of telomerase by human papillomaviruses.

The E6 oncoprotein of human papillomaviruses (HPVs) induces telomerase activity in primary human epithelial cells. This activity is dependent on association of E6 with E6AP, a cellular ubiquitin ligase. E6 activates the transcription of hTERT, the catalytic subunit of telomerase. E boxes near the start of hTERT transcription are required for E6; however, acetylated histones are only present in the E6 cells. We identified two isoforms of NFX1, a new binding partner of E6/E6AP. The NFX1- 91 isoform binds to an X-box motif located adjacent to the proximal E box, binds Sin3A and HDACs, repressing hTERT transcription. It preferentially binds E6/E6AP and is targeted for ubiquitin-mediated degradation. The NFX1-123 isoform has the opposite activity, increasing hTERT transcription or translation. This is the first example of viral oncoproteins disrupting regulation of telomerase, a critical event in tumorigenesis.

E box-dependent activation of telomerase by human papillomavirus type 16 E6 does not require induction of c-myc.

Human papillomavirus type 16 (HPV-16) E6 activates telomerase specifically in epithelial cells. The oncogene c-myc has also been shown to activate telomerase in several cell types. Here we show that while both HPV-16 E6 and c-myc require intact E boxes to transactivate the hTERT promoter, E6 does not induce hTERT transcription simply by inducing expression of c-myc. Moreover, hTERT transactivation by HPV-16 E6 correlates with its ability to bind the cellular E6-associated protein (E6AP), suggesting that E6 and E6AP may target a regulator of hTERT expression.

Promoter cloning in the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans is a highly radiation-resistant bacterium that is classed in a major subbranch of the bacterial domain. Since very little is known about gene expression in this bacterium, an initial study of promoters was undertaken. In order to isolate promoters and study promoter function, a series of integrative vectors for stable chromosomal insertion in D. radiodurans were developed. These vectors are based on Escherichia coli replicons that are unable to replicate autonomously in D. radiodurans and carry homologous sequences for replacement recombination in the D. radiodurans chromosome. The resulting integration vectors were used to study expression of reporter genes fused to a number of putative promoters that were amplified from the D. radiodurans R1 genome. Further analysis of these and other putative promoters was performed by Northern hybridization and primer extension experiments. In contrast to previous reports, the -10 and -35 regions of these promoters resembled the sigma(70) consensus sequence of E. coli.

The G(2) checkpoint is maintained by redundant pathways.

While p53 activity is critical for a DNA damage-induced G(1) checkpoint, its role in the G(2) checkpoint has not been compelling because cells lacking p53 retain the ability to arrest in G(2) following DNA damage. Comparison between normal human foreskin fibroblasts (HFFs) and HFFs in which p53 was eliminated by transduction with human papillomavirus type 16 E6 showed that treatment with adriamycin initiated arrest in G(2) with active cyclin B/CDC2 kinase, regardless of p53 status. Both E6-transduced HFFs and control (LXSN)-transduced cells maintained a prolonged arrest in G(2); however cells with functional p53 extinguished cyclin B-associated kinase activity. Down regulation was mediated by p53-dependent transcriptional repression of the CDC2 and cyclin B promoters. In contrast, cells lacking p53 showed a prolonged G(2) arrest despite high levels of cyclin B/CDC2 kinase activity, at least some of which translocated into the nucleus. Furthermore, the G(2) checkpoint became attenuated as p53-deficient cells aged in culture. Thus, at late passage, E6-transduced HFFs entered mitosis following DNA damage, whereas the age-matched parental HFFs sustained a G(2) arrest. These results indicate that normal cells have p53-independent pathways to maintain DNA damage-induced G(2) arrest, which may be augmented by p53-dependent functions, and that cells lacking p53 are at greater risk of losing the pathway that protects against aneuploidy.