Epidermal Loss of RORα Enhances Skin Inflammation in a MC903-Induced Mouse Model of Atopic Dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease featuring skin barrier dysfunction and immune dysregulation. Previously, we reported that the retinoid-related orphan nuclear receptor RORα was highly expressed in the epidermis of normal skin. We also found that it positively regulated the expression of differentiation markers and skin barrier-related genes in human keratinocytes. In contrast, epidermal RORα expression was downregulated in the skin lesions of several inflammatory skin diseases, including AD. In this study, we generated mouse strains with epidermis-specific Rora ablation to understand the roles of epidermal RORα in regulating AD pathogenesis. Although Rora deficiency did not cause overt macroscopic skin abnormalities at the steady state, it greatly amplified MC903-elicited AD-like symptoms by intensifying skin scaliness, increasing epidermal hyperproliferation and barrier impairment, and elevating dermal immune infiltrates, proinflammatory cytokines, and chemokines. Despite the normal appearance at the steady state, Rora-deficient skin showed microscopic abnormalities, including mild epidermal hyperplasia, increased TEWL, and elevated mRNA expression of Krt16, Sprr2a, and Tslp genes, indicating subclinical impairment of epidermal barrier functions. Our results substantiate the importance of epidermal RORα in partially suppressing AD development by maintaining normal keratinocyte differentiation and skin barrier function.

Loss of haspin suppresses cancer cell proliferation by interfering with cell cycle progression at multiple stages.

Our recent studies have shown that haspin, a protein kinase imperative for mitosis, is engaged in the interphase progression of HeLa and U2OS cancer cells. In this investigation, we employed the Fucci reporter system and time-lapse imaging to examine the impact of haspin gene silencing on cell cycle progressions at a single-cell level. We found that the loss of haspin induced multiple cell cycle defects. Specifically, the S/G2 duration was greatly prolonged by haspin gene depletion or inhibition in synchronous HeLa cells. Haspin gene depletion in asynchronous HeLa and U2OS cells led to a similarly protracted S/G2 phase, followed by mitotic cell death or postmitotic G1 arrest. In addition, haspin deficiency resulted in robust induction of the p21CIP1/WAF1 checkpoint protein, a target of the p53 activation. Also, co-depleting haspin with either p21 or p53 could rescue U2OS cells from postmitotic G1 arrest and partially restore their proliferation. These results substantiate the haspin's capacity to regulate interphase and mitotic progression, offering a broader antiproliferative potential of haspin loss in cancer cells.

Haspin inhibition delays cell cycle progression through interphase in cancer cells.

Haspin (Haploid Germ Cell-Specific Nuclear Protein Kinase) is a serine/threonine kinase pertinent to normal mitosis progression and mitotic phosphorylation of histone H3 at threonine 3 in mammalian cells. Different classes of small molecule inhibitors of haspin have been developed and utilized to investigate its mitotic functions. We report herein that applying haspin inhibitor CHR-6494 or 5-ITu at the G1/S boundary could delay mitotic entry in synchronized HeLa and U2OS cells, respectively, following an extended G2 or the S phase. Moreover, late application of haspin inhibitors at S/G2 boundary is sufficient to delay mitotic onset in both cell lines, thereby, indicating a direct effect of haspin on G2/M transition. A prolonged interphase duration is also observed with knockdown of haspin expression in synchronized and asynchronous cells. These results suggest that haspin can regulate cell cycle progression at multiple stages at both interphase and mitosis.

Vesicular translocation of PARP-1 to cytoplasm causes ADP-ribosylation and disassembly of vimentin filaments during microglia activation induced by LPS.

ADP-ribosylation plays a significant role in various biological processes including genomic stability maintenance, transcriptional regulation, energy metabolism, and cell death. Using macrodomain pull-down assay with microglia lysates and MALDI-TOF-MS analysis, we identified vimentin as a major protein highly ADP-ribosylated by the poly(ADP-ribose) polymerases-1 (PARP-1) in response to LPS. ABT-888, a potent inhibitor of PARP-1/2 blocks the disassembly and ADP-ribosylation of vimentin. PARP-1 is a highly abundant nuclear protein. Its nuclear functions in repairing DNA damages induced by various stress signals, such as inflammatory stresses, have been well studied. In contrast, limited studies have been done on the cytoplasmic role(s) of PARP-1. Our study focuses on the cytoplasmic role of PARP-1 during microglia activation. Using immunofluorescence microscopy and Western blotting, we showed that a significant amount of PARP-1 is present in the cytosol of microglia cells stimulated and activated by LPS. Live cell imaging showed the translocation of nuclear PARP-1-EGFP to the cytoplasm in vesicular structures upon LPS stimulation. ABT-888 and U0126 can block this translocation. Immunofluorescence staining with various organelle marker antibodies revealed that PARP-1 vesicles show colocalization with Lamin A/C, suggesting they might be derived from the nuclear envelope through nuclear envelope budding. In conclusion, we demonstrated that PARP-1 is translocated from the nucleus to cytoplasm via vesicles upon LPS stimulation and that cytoplasmic PARP-1 causes ADP-ribosylation and disassembly of vimentin filaments during microglia activation induced by LPS.