Ubiquitin-Dependent Proteolysis of Cyclin D1 Is Associated with Coxsackievirus-Induced Cell Growth Arrest

Coxsackievirus group B3 (CVB3) replication is influenced by host cell cycle status. However, the effect of CVB3 infection on cell cycle regulation and the mechanisms involved are not precisely defined. In this study, we examined cell cycle progression and regulation when the infection was initiated in late G(1) phase of the cell cycle. Analysis of cellular DNA synthesis in infected cells by thymidine incorporation assays showed a significant reduction in [(3)H]thymidine uptake compared to that of sham-infected cells. To further clarify the effects of CVB3 on the host cell cycle, we examined the cell cycle regulatory proteins involved in G(1) progression and G(1)/S transition. Infection resulted in dephosphorylation of retinoblastoma protein and reduced G(1) cyclin-dependent kinase activities, accompanied by decreased levels of G(1) cyclin protein expression (cyclin D1 and cyclin E). We further investigated the mechanisms by which CVB3 infection down-regulates cyclin D1 expression. Northern blotting showed that cyclin D1 mRNA levels were modestly increased following CVB3 infection, suggesting that cyclin D1 regulation occurs by a posttranscriptional mechanism. Viral infection resulted in only a 20 to 30% inhibition of cyclin D1 protein synthesis 3 h postinfection. However, the proteasome inhibitors MG132 and lactacystin prevent CVB3-induced cyclin D1 reduction, indicating that CVB3-induced down-regulation of cyclin D1 is facilitated by ubiquitin-proteasome proteolysis. Finally, using GSK3β pathway inhibitors, we showed that the reduction of cyclin D1 is GSK3β independent. Taken together, our results demonstrate that CVB3 infection disrupts host cell homeostasis by blocking the cell cycle at the G(1)/S boundary and induces cell cycle arrest in part through an increase in ubiquitin-dependent proteolysis of cyclin D1

Expression of SMA inhibits SMC proliferation and migration but does not affect activation of MAPKs.

<p>(A) Proliferation of GFP-controls or SMCs overexpressing SMA was determined by MTT assays at various time points after stimulation with 10% FBS. Data (OD₅₆₀ measurements) are presented as mean +/- S.D. of 6 independent experiments *P< 0.001. (B) Cell migration in response to 10% FBS was measured by scratch assays. Hydroxyurea at 5 mmol/L was added to prevent proliferation. Data (percent area covered by cells 17 hours after wounding) are presented as mean +/- S.D. of 6 independent experiments. *P<0.001. Representative micrographs for GFP-controls (co) and SMA-SMCs (SMA) are shown at time 0 right after wounding and at the end of the experiment. (C) Serum-starved cells (GFP-controls and SMA-SMCs) were stimulated with 10% FBS and harvested at the time points indicated. Cell extracts were subjected to Western blot analysis and blots probed with antibodies to recognize the phosphorylated forms of p44/p42 ERK, p38 MAPK or p54/p46 JNK. In all cases, HSC70 was used as a loading control. For each phospho-antibody, a representative result from three independent experiments is shown. The band below phospho-p46 JNK is considered a singularly phosphorylated form that does not shift, or a breakdown product.</p

SMA inhibits serum-induced activation of Rac.

<p>Quiescent cells (GFP-controls or SMCs overexpressing SMA) were stimulated with 10% FBS for 1 and 5 min. Cell lysates were prepared and the amount of GTP-bound Rac (A) and Rho (B) determined by ELISA. All assays were performed in triplicates with two independently prepared cell batches for each condition. Data (OD₄₉₀ measurements) are presented as mean +/- S.D. *P<0.001, n.s. = non-significant. Inserts show Western blots for total Rac and Rho protein in extracts of GFP-controls and SMA-SMCs. HSC-70 was used to confirm equal protein loading.</p

Expression of a constitutively active Rac1 mutant counteracts the inhibitory effects of SMA on cell migration and growth.

<p>Controls or SMCs overexpressing SMA were transfected with adenovirus to express lacZ (Ad-lacZ) or constitutively active V12rac1 (Ad-rac). All assays were performed in triplicates. (A) Proliferation of cells was determined by MTT assays at day 1 and day 5 after stimulation with 10% FBS. Data (OD₅₆₀ measurements) are presented as mean +/- S.D. (B) Cell migration in response to 10% FBS was measured by scratch assays. Hydroxyurea at 5 mmol/L was added to prevent proliferation. Data (percent area covered by cells 17 hours after wounding) are presented as mean +/- S.D.</p

Visualization of actin fibers and focal adhesion sites in SMCs overexpressing SMA.

<p>SMCs were transfected with bicistronic lenti virus expressing GFP alone (co, GFP-control), or in addition VSVG-tagged SMA (SMA). (A) Overexpression of SMA was confirmed by Western blot analysis of cell extracts using antibodies against SMA or VSVG. Staining for β-tubulin was included as loading control. Two independent batches of cells were prepared for controls and SMA overexpression. Note the slight up-shift of the SMA band in SMA-SMC-extracts due to the VSVG-tag. (B) GFP-controls or SMA-SMCs were plated onto fibronectin-coated coverslips and stained with phalloidin for F-actin or with antibody for vinculin. A typical micrograph for each condition including a magnification of an area of interest is shown. Bars equal 50 μm.</p

Knock-down of SMA expression increases the activation of Rac.

<p>SMCs were transfected with non-specific, GC-matched siRNA (control) or siRNA targeting SMA (SMA). (A) Expression of SMA was determined by qPCR measurements and normalized to GAPDH expression. Data (mean + S.D.) from 8 independent transfection experiments are shown. Insert: Western blot analysis of SMA protein expression. The blot was reprobed with beta-actin antibody as a loading control. (B, C) Quiescent controls or cells transfected with SMA-siRNA were stimulated with 10% FBS for 1 and 5 min. Cell lysates were prepared and the amount of GTP-bound Rac (B) and RhoA (C) determined by ELISA following the manufacturer’s instructions. Assays were performed in triplicates with two independent cell batches for each condition. Data (OD490 measurements) are presented as mean +/- S.D. *P<0.001, n.s. = non-significant.</p