Tumor cell cycle arrest induced by shear stress: Roles of integrins and Smad

Abstract

Interstitial flow in and around tumor tissue affects the mechanical microenvironment to modulate tumor cell growth and metastasis. We investigated the roles of flow-induced shear stress in modulating cell cycle distribution in four tumor cell lines and the underlying mechanisms. In all four cell lines, incubation under static conditions for 24 or 48 h led to G(0)/G(1) arrest; in contrast, shear stress (12 dynes/cm(2)) induced G(2)/M arrest. The molecular basis of the shear effect was analyzed, and the presentation on molecular mechanism is focused on human MG63 osteosarcoma cells. Shear stress induced increased expressions of cyclin B1 and p21(CIP1) and decreased expressions of cyclins A, D1, and E, cyclin-dependent protein kinases (Cdk)-1, -2, -4, and -6, and p27(KIP1) as well as a decrease in Cdk1 activity. Using specific antibodies and small interfering RNA, we found that the shear-induced G(2)/M arrest and corresponding changes in G(2)/M regulatory protein expression and activity were mediated by alpha(v)beta(3) and beta(1) integrins through bone morphogenetic protein receptor type IA-specific Smad1 and Smad5. Shear stress also down-regulated runt-related transcription factor 2 (Runx2) binding activity and osteocalcin and alkaline phosphatase expressions in MG63 cells; these responses were mediated by alpha(v)beta(3) and beta(1) integrins through Smad5. Our findings provide insights into the mechanism by which shear stress induces G(2)/M arrest in tumor cells and inhibits cell differentiation and demonstrate the importance of mechanical microenvironment in modulating molecular signaling, gene expression, cell cycle, and functions in tumor cells.

Fig. 1.

Shear stress regulates expressions of cell cycle regulatory proteins in MG63 cells. MG63 cells were kept as controls or subjected to shear stress (12 dynes/cm²) for 24 and 48 h. Expression of cell cycle regulatory proteins was determined by Western blot analysis. Data are means ± SEM from three independent experiments. *, P < 0.05 vs. static control cells.

Fig. 2.

Shear stress induces sustained phosphorylation of Smad1/5 in MG63 cells through BMPRIA. (A) MG63 cells were kept as controls or subjected to shear stress (12 dynes/cm²) for 10 min, 30 min, 1 h, 3 h, 6 h, and 24 h, and their Smad 1/5 phosphorylation was determined by Western blot analysis. (B) MG63 cells were exposed to shear stress of 2, 12, and 20 dynes/cm² for 30 min. (C) MG63 cells were kept as controls (−) or pretreated with Noggin (100 ng/ml) for 1 h (+N) and then subjected to flow or BMP-4 (100 ng/ml) for 30 min. (D) MG63 cells were transfected with siRNAs at various concentrations (5, 15, 30, and 40 nM), and their BMPRIA and BMPRIB protein expressions were examined by Western blot analysis. (E) MG63 cells were transfected with control siRNA (siCL) or specific siRNA of BMPRIA (siRIA) or BMPRIB (siRIB) (40 nM each) for 48 h and then kept as controls (C) or exposed to flow (S) for 30 min. Data in A and B are means ± SEM from three independent experiments and presented as percentage changes in band density from control cells normalized to Smad1/5 protein levels. The results in C–E are representative of triplicate experiments with similar results. *, P < 0.05 vs. static control cells.

Fig. 3.

Shear-induced G₂/M arrest in MG63 cells is mediated by Smad1/5. (A) MG63 cells were transfected with Smad1- or Smad5-specific siRNA at 5, 15, 30, or 40 nM for 48 h, and Smad1 and Smad5 protein expressions were determined by Western blot analysis. Results are representative of triplicate experiments with similar results. (B) MG63 cells were transfected with control siRNA (siCL) or specific siRNA of Smad1 (siSmad1) or Smad5 (siSamd5) (40 nM each) and then kept under static conditions (C) or exposed to flow (S) for 48 h. The expression of cell cycle regulatory proteins was determined by Western blot analysis. Data are means ± SEM from three independent experiments and are presented as percentage changes in band density from control cells normalized to Smad1/5 protein levels. #, P < 0.05 vs. cells transfected with control siRNA.

Fig. 4.

Shear-induced Smad1/5 activations are mediated by α_vβ₃ and β₁ integrins in MG63 cells. (A) MG63 cells were pretreated with RGDS peptides (500 μg/ml) or specific antibodies against β₁ (Anti-β₁) or α_vβ₃ (Anti-α_vβ₃) for 2 h (10 μg/ml each) and kept under static conditions (C) or subjected to shear stress (12 dynes/cm²) (S) for 30 min. As controls, the cells were pretreated with RGES (500 μg/ml) and nonspecific control IgG (10 μg/ml). (B) MG63 cells were transfected with control siRNA (siCL) or specific siRNA of α_v (siα_v), β₁ (siβ₁), or β₃ (siβ₃) (40 nM each) for 48 h before exposure to flow. (C) MG63 cells were transfected with specific siRNAs of integrins at 5, 15, 30, or 40 nM, and their integrin protein expressions were examined. (D) MG63 cells were transfected with 3 μg of Shc-SH2 (mShc), FAK(F397Y) (mFAK), or pcDNA3 empty vector for 48 h before exposure to flow (S). Results are representative of triplicate experiments with similar results.

Fig. 5.

MG63 cell differentiation and Runx2 binding activity in the nucleus were inhibited by shear stress acting through α_vβ₃ and β₁ integrins and Smad5. (A–C) MG63 cells were kept as controls (C) or subjected to shear stress (12 dynes/cm²) for 30 min to 24 h, as indicated. (B, D–F) The cells were transfected with empty vector control PSRα or OCN-Luc (1 μg/ml each) (B), transfected with siRNA (siCL for control, or specific siRNA of Smad1 or Smad5) (40 nM each) (D and E) for 48 h, or pretreated with control IgG or a specific antibody against α_vβ₃ or β₁ (10 μg/ml each) for 2 h (F) and then were kept under static conditions or subjected to shear stress for 6 and 24 h (B) or 24 h (D–F). The OCN and ALP mRNA expressions (A, D, and E), OCN promoter and ALP activities (B), and Runx2-DNA binding activity (C and E) were determined by real-time PCR, luciferase and ALP activity assays, and EMSA, respectively. In C, total nuclear extracts of cells and ³²P-labeled oligonucleotides containing human OCN Runx2-binding sites were used. Nuclear extracts were preincubated with 20-fold excess unlabeled oligonucleotides (+20×). As positive controls, MG63 cells were treated with BMP-4 (100 ng/ml) for 6 and 24 h (C). Nuclear extracts preincubated with the Runx2 antibody (+Ab) show a super shift band (SH). Results in A, B, D, and F are means ± SEM from three to four independent experiments. Results in C and E are representative of two or three independent experiments with similar results. *, P < 0.05 vs. static control cells (A and B); #, P < 0.05 vs. control treatments (D and F).

Fig. 6.

Schematic representation of the signaling pathways regulating cell cycle and differentiation in tumor cells in response to shear stress. ↑, up-regulation by shear; ↓, down-regulation by shear; dotted double-arrow line represents the interaction pathway that has not been defined.