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Deficiency of the 15-kDa selenoprotein led to cytoskeleton remodeling and non-apoptotic membrane blebbing through a RhoA/ROCK pathway.

ABSTRACT: The 15-kDa selenoprotein (Sep15) has been implicated in etiology of some types of cancer. Herein, inducible RNAi cell lines were established and cell morphology and motility were analyzed. The majority of Sep15-deficient cells (>95%) formed membrane blebs in a dynamic manner. Blebbing cells transformed cell morphology from a normal flat spindle shape to a spherical morphology. In blebbing cells, actin fibers moved to the cell periphery, covering and obscuring visualization of ?-tubulin. Bleb formation was suppressed by the inhibitors of Rho-associated protein kinase (ROCK), RhoA or myosin light chain (MLC), restoring blebbing cells to wild-type morphology. RhoA activation and phosphorylation of myosin phosphatase target subunit 1 was induced by Sep15 knockdown. Sep15-deficient cells were non-apoptotic, and displayed a distinct relative localization of F-actin and ?-tubulin from typical apoptotic blebbing cells. Our data suggest that Sep15 in Chang liver cells regulates the pathway that antagonizes RhoA/ROCK/MLC-dependent non-apoptotic bleb formation.

SUBMITTER: Bang J

PROVIDER: S-EPMC4758352 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Deficiency of the 15-kDa selenoprotein led to cytoskeleton remodeling and non-apoptotic membrane blebbing through a RhoA/ROCK pathway.

Bang Jeyoung J Jang Mihyun M Huh Jang Hoe JH Na Ji-Woon JW Shim Myoungsup M Carlson Bradley A BA Tobe Ryuta R Tsuji Petra A PA Gladyshev Vadim N VN Hatfield Dolph L DL Lee Byeong Jae BJ

Biochemical and biophysical research communications 20141218 4

The 15-kDa selenoprotein (Sep15) has been implicated in etiology of some types of cancer. Herein, inducible RNAi cell lines were established and cell morphology and motility were analyzed. The majority of Sep15-deficient cells (>95%) formed membrane blebs in a dynamic manner. Blebbing cells transformed cell morphology from a normal flat spindle shape to a spherical morphology. In blebbing cells, actin fibers moved to the cell periphery, covering and obscuring visualization of α-tubulin. Bleb for ...[more]

PMID: 25529450

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Project description:Selenium has cancer preventive activity that is mediated, in part, through selenoproteins. The role of the 15 kDa selenoprotein (Sep15) in colon cancer was assessed by preparing and using mouse colon CT26 cells stably transfected with shRNA constructs targeting Sep15. Metabolic 75Se-labeling and Northern and Western blot analyses revealed that more than 90% of Sep15 was knocked down. Growth of the resulting Sep15-deficient CT26 cells was reduced (p<0.01) and cells formed significantly (p<0.001) fewer colonies in soft agar compared to control CT26 cells. Whereas most (14/15) BALB/c mice injected with control cells developed tumors, few (3/30) mice injected with Sep15 knockdown cells developed tumors (p<0.0001). The ability to form pulmonary metastases had similar results. Mice injected with the plasmid-transfected control cells had >250 lung metastases/mouse; however, mice injected with the Sep15 knockdown cells only had 7.8 +/- 5.4 metastases. To investigate molecular targets affected by Sep15 status, gene expression patterns between control and knockdown CT26 cells were compared. Ingenuity Pathways Analysis was used to analyze the 1045 genes that were significantly (p<0.001) affected by Sep15 deficiency. The highest scored biological functions were cancer and cellular growth and proliferation. Consistent with these observations, subsequent analyses revealed a G2/M cell cycle arrest in Sep15 CT26 knockdown cells. In contrast, to CT26 cells Sep15 knockdown in Lewis Lung Carcinoma (LLC1) cells did not affect anchorage-dependent or M-bM-^@M-^Sindependent cell growth. These data suggest tissue specificity in the cancer protective effects of Sep15 knockdown, which are mediated, at least in part, by influencing the cell cycle. mRNA was isolated from plasmid-transfected control and shSep15 knockdown CT26 cells (three replicates of each). Microarray analysis was performed on Affymetrix Mouse 430_2 gene chips. Three arrays were analyzed from different mRNA samples for each construct.

Project description:Osteoarthritis (OA) is a progressive joint disorder, which is principally characterized by the degeneration and destruction of articular cartilage. The cytoskeleton is a vital structure that maintains the morphology and function of chondrocytes, and its destruction is a crucial risk factor leading to chondrocyte degeneration and OA. Hyaluronan synthase‑2 (HAS‑2) is a key enzyme in synthesizing hyaluronic acid (HA) in vivo. The synthesis of high molecular weight HA catalyzed by HAS‑2 serves a vital role in joint movement and homeostasis; however, it is unclear what important role HAS‑2 plays in maintaining chondrocyte cytoskeleton morphology and in cartilage degeneration. The present study downregulated the expression of HAS‑2 by employing 4‑methylumbelliferone (4‑MU) and RNA interference. In vitro experiments, including reverse transcription‑quantitative PCR, western blotting, laser scanning confocal microscopy and flow cytometry were subsequently performed. The results revealed that downregulation of HAS‑2 could activate the RhoA/ROCK signaling pathway, cause morphological abnormalities, decrease expression of the chondrocyte cytoskeleton proteins and promote chondrocyte apoptosis. In vivo experiments, including immunohistochemistry and Mankin's scoring, were performed to verify the effect of HAS‑2 on the chondrocyte cytoskeleton, and it was revealed that inhibition of HAS‑2 could cause cartilage degeneration. In conclusion, the present results revealed that downregulation of HAS‑2 could activate the RhoA/ROCK pathway, cause abnormal morphology and decrease chondrocyte cytoskeleton protein expression, leading to changes in the signal transduction and biomechanical properties of chondrocytes, promotion of chondrocyte apoptosis and the induction of cartilage degeneration. Moreover, the clinical application of 4‑MU may cause cartilage degeneration. Therefore, targeting HAS‑2 may provide a novel therapeutic strategy for delaying chondrocyte degeneration, and the early prevention and treatment of OA.

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Deficiency of the 15-kDa selenoprotein led to cytoskeleton remodeling and non-apoptotic membrane blebbing through a RhoA/ROCK pathway.

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Deficiency of the 15-kDa selenoprotein led to cytoskeleton remodeling and non-apoptotic membrane blebbing through a RhoA/ROCK pathway.

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