Pei-Ling Lee

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

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 G0/G1 arrest; in contrast, shear stress (12 dynes/cm2) induced G2/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 p21CIP1 and decreased expressions of cyclins A, D1, and E, cyclin-dependent protein kinases (Cdk)-1, -2, -4, and -6, and p27KIP1 as well as a decrease in Cdk1 activity. Using specific antibodies and small interfering RNA, we found that the shear-induced G2/M arrest and corresponding changes in G2/M regulatory protein expression and activity were mediated by αvβ3 and β1 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 αvβ3 and β1 integrins through Smad5. Our findings provide insights into the mechanism by which shear stress induces G2/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.

Analysis of the effect of disturbed flow on monocytic adhesion to endothelial cells

The preferential adhesion of monocytes to vascular endothelial cells (ECs) at regions near branches and curvatures of the arterial tree, where flow is disturbed, suggests that hemodynamic conditions play significant roles in monocyte adhesion. The present study aims to elucidate the effects of disturbed flow on monocyte adhesion to ECs and the adhesive properties of ECs. We applied, for the first time, the micron-resolution particle image velocimetry (microPIV) technique to analyze the characteristics of the disturbed flow produced in our vertical-step flow (VSF) chamber. The results demonstrated the existence of a higher near-wall concentration and a longer residence time of the monocytic analog THP-1 cells near the step and the reattachment point. THP-1 cells showed prominent adhesion to ECs pretreated with TNFalpha in the regions near the step and the reattachment point, but they showed virtually no adhesion to un-stimulated ECs. Pre-incubation of the TNFalpha-treated ECs with antibodies against intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin inhibited the THP-1 adhesion; the maximal inhibition was observed with a combination of these antibodies. Pre-exposure of ECs to disturbed flow in VSF for 24 h led to significant increases in their surface expressions of ICAM-1 and E-selectin, but not VCAM-1, and in the adhesion of THP-1 cells. Our findings demonstrate the importance of complex flow environment in modulating the adhesive properties of vascular endothelium and consequently monocyte adhesion in regions of prevalence of atherosclerotic lesions.

Integrin-mediated expression of bone formation-related genes in osteoblast-like cells in response to fluid shear stress: roles of extracellular matrix, Shc, and mitogen-activated protein kinase.

Integrins play significant roles in mechanical responses of cells on extracellular matrix (ECM). We studied the roles of integrins and ECM proteins (fibronectin [FN], type I collagen [COL1], and laminin [LM]) in shear‐mediated signaling and the expression of bone formation‐related genes (early growth response‐1 [Egr‐1], c‐fos, cyclooxygenase‐2 [Cox‐2], and osteopontin [OPN]) in human osteosarcoma MG63 cells. MG63 cells on FN, COL1, and LM were kept as controls or subjected to shear stress (12 dynes/cm2), and the association of αvβ3 and β1 integrins with Shc, phosphorylation of mitogen‐activated protein kinases (MAPKs, i.e., extracellular signal‐regulated kinase [ERK], c‐jun‐NH2‐terminal kinase [JNK], and p38), and expressions of Egr‐1, c‐fos, Cox‐2, and OPN were determined. In MG63 cells, shear stress induces sustained associations of αvβ3 and β1 with Shc when seeded on FN, but sustained associations of only β1 with Shc when seeded on COL1/LM. Shear inductions of MAPKs and bone formation‐related genes were sustained (24 h) in cells on FN, but some of these responses were transient in cells on COL1/LM. The shear activations of ERK, JNK, and p38 were mediated by integrins and Shc, and these pathways differentially modulated the downstream bone formation‐related gene expression. Our findings showed that β1 integrin plays predominant roles for shear‐induced signaling and gene expression in osteoblast‐like MG63 cells on FN, COL1, and LM and that αvβ3 also plays significant roles for such responses in cells on FN. The β1/Shc association leads to the activation of ERK, which is critical for shear induction of bone formation‐related genes in osteoblast‐like cells.

Shear Stress Inhibits Smooth Muscle Cell–Induced Inflammatory Gene Expression in Endothelial Cells Role of NF-κB

Objectives—Vascular endothelial cells (ECs) are influenced by shear stress and neighboring smooth muscle cells (SMCs). We investigated the inflammation-relevant gene expression in EC/SMC cocultures under static condition and in response to shear stress. Materials and Methods—Under static condition, DNA microarrays and reverse-transcription polymerase chain reaction identified 23 inflammation-relevant genes in ECs whose expression was significantly affected by coculture with SMCs, with 18 upregulated and 5 downregulated. Application of shear stress (12 dynes/cm2) to the EC side of the coculture for 6 hours inhibited most of the proinflammatory gene expressions in ECs induced by coculture with SMCs. Inhibition of nuclear factor-&kgr;B (NF-&kgr;B) activation by the p65-antisense, lactacystin, and N-acetyl-cysteine blocked the coculture-induced EC expression of proinflammatory genes, indicating that the NF-&kgr;B binding sites in the promoters of these genes play a significant role in their expression as a result of coculture with SMCs. Chromatin immunoprecipitation assays demonstrated the in vivo regulation of NF-&kgr;B recruitment to selected target promoters. Shear stress inhibited the SMC coculture-induced NF-&kgr;B activation in ECs and monocytic THP-1 cell adhesion to ECs. Conclusions—Our findings suggest that shear stress plays an inhibitory role in the proinflammatory gene expression in ECs located in close proximity to SMCs.

Mechanical regulation of cancer cell apoptosis and autophagy: roles of bone morphogenetic protein receptor, Smad1/5, and p38 MAPK.

Mechanical forces induced by interstitial fluid flow in and surrounding tissues and by blood/lymphatic flow in vessels may modulate cancer cell invasion and metastasis and anticancer drug delivery. Our previous study demonstrated that laminar flow-induced shear stress induces G2/M arrest in tumor cells. However, whether shear stress modulates final cell fate remains unclear. In this study, we investigated the role of flow-induced shear stress in modulating the survival of four human tumor cell lines, i.e., Hep3B hepatocarcinoma cells, MG63 osteosarcoma cells, SCC25 oral squamous carcinoma cells, and A549 carcinomic alveolar basal epithelial cells. Laminar shear stress (LSS) ranging from 0.5 to 12dyn/cm(2) induced death of these four tumor cell lines. In contrast to LSS at 0.5dyn/cm(2), oscillatory shear stress (OSS) at 0.5±4dyn/cm(2) cannot induce cancer cell death. Both LSS and OSS had no effect on human normal hepatocyte, lung epithelial, and endothelial cells. Application of LSS to these four cell lines increased the percentage of cells stained positively for annexin V-FITC, with up-regulations of cleaved caspase-8, -9, and -3, and PARP. In addition, LSS also induced Hep3B cell autophagy, as detected by acidic vesicular organelle formation, LC3B transformation, and p62/SQSTM1 degradation. By transfecting with small interfering RNA, we found that the shear-induced apoptosis and autophagy are mediated by bone morphogenetic protein receptor type (BMPR)-IB, BMPR-specific Smad1 and Smad5, and p38 mitogen-activated protein kinase in Hep3B cells. Our findings provide insights into the molecular mechanisms by which shear stress induces apoptosis and autophagy in tumor cells.

BMP receptor-integrin interaction mediates responses of vascular endothelial Smad1/5 and proliferation to disturbed flow.

Vascular endothelial cells (ECs) are constantly exposed to blood flow‐induced shear stress. Our previous study demonstrated that disturbed flow with low and oscillatory shear stress (OSS) induces bone morphogenetic protein receptor (BMPR)‐specific Smad1/5 activation in ECs, but the underlying mechanisms and the in vivo functional role of Smad1/5 remain unclear.

Estrogen Augments Shear Stress–Induced Signaling and Gene Expression in Osteoblast-like Cells via Estrogen Receptor–Mediated Expression of β1-Integrin

Estrogen and mechanical forces are positive regulators for osteoblast proliferation and bone formation. We investigated the synergistic effect of estrogen and flow‐induced shear stress on signal transduction and gene expression in human osetoblast‐like MG63 cells and primary osteoblasts (HOBs) using activations of extracellular signal‐regulated kinase (ERK) and p38 mitogen‐activated protein kinase (MAPK) and expressions of c‐fos and cyclooxygenase‐2 (I) as readouts. Estrogen (17β‐estradiol, 10 nM) and shear stress (12 dyn/cm2) alone induced transient phosphorylations of ERK and p38 MAPK in MG63 cells. Pretreating MG63 cells with 17β‐estradiol for 6 hours before shearing augmented these shear‐induced MAPK phosphorylations. Western blot and flow cytometric analyses showed that treating MG63 cells with 17β‐estradiol for 6 hrs induced their β1‐integrin expression. This estrogen‐induction of β1‐integrin was inhibited by pretreating the cells with a specific antagonist of estrogen receptor ICI 182,780. Both 17β‐estradiol and shear stress alone induced c‐fos and Cox‐2 gene expressions in MG63 cells. Pretreating MG63 cells with 17β‐estradiol for 6 hrs augmented the shear‐induced c‐fos and Cox‐2 expressions. The augmented effects of 17β‐estradiol on shear‐induced MAPK phosphorylations and c‐fos and Cox‐2 expressions were inhibited by pretreating the cells with ICI 182,780 or transfecting the cells with β1‐specific small interfering RNA. Similar results on the augmented effect of estrogen on shear‐induced signaling and gene expression were obtained with HOBs. Our findings provide insights into the mechanism by which estrogen augments shear stress responsiveness of signal transduction and gene expression in bone cells via estrogen receptor–mediated increases in β1‐integrin expression.

MicroRNA-10a is crucial for endothelial response to different flow patterns via interaction of retinoid acid receptors and histone deacetylases

Significance This study demonstrates that hormone receptor RARα plays a vital role in the selective activation of proinflammatory and anti-inflammatory signaling to modulate the miR-10a/GATA6/VCAM-1 cascade in endothelial cells in response to proatherogenic oscillatory shear stress (OS) vs. atheroprotective pulsatile shear stress (PS). HDAC-3/5/7 and RXRα are induced by OS and PS to serve as mechanosensitive “repressors” and “enhancers,” respectively, to associate with RARα to modulate its binding to RA-responsive element (RARE) to switch miR-10a expression. Our findings provide insight into the relationship between two different epigenetic factors (HDACs and miRs) and hormone receptors (RARα and RXRα) in the regulation of endothelial functions and elucidate new mechanisms of hemodynamic-based pathophysiology of the atherosclerotic vascular wall. Histone deacetylases (HDACs) and microRNAs (miRs) have emerged as two important epigenetic factors in the regulation of vascular physiology. This study aimed to elucidate the relationship between HDACs and miRs in the hemodynamic modulation of endothelial cell (EC) dysfunction. We found that miR-10a has the lowest expression among all examined shear-responsive miRs in ECs under oscillatory shear stress (OS), and a relatively high expression under pulsatile shear stress (PS). PS and OS alter EC miR-10a expression to regulate the expression of its direct target GATA6 and downstream vascular cell adhesion molecule (VCAM)-1. PS induces the expression, nuclear accumulation, and association of retinoid acid receptor-α (RARα) and retinoid X receptor-α (RXRα). RARα and RXRα serve as a “director” and an “enhancer,” respectively, to enhance RARα binding to RA-responsive element (RARE) and hence miR-10a expression, thus down-regulating GATA6/VCAM-1 signaling in ECs. In contrast, OS induces associations of “repressors” HDAC-3/5/7 with RARα to inhibit the RARα-directed miR-10a signaling. The flow-mediated miR-10a expression is regulated by Krüppel-like factor 2 through modulation in RARα–RARE binding, with the consequent regulation in GATA6/VCAM-1 in ECs. These results are confirmed in vivo by en face staining on the aortic arch vs. the straight thoracic aorta of rats. Our findings identify a mechanism by which HDACs and RXRα modulate the hormone receptor RARα to switch miR-10a expression and hence the proinflammatory vs. anti-inflammatory responses of vascular endothelium under different hemodynamic forces.

Differential regulations of fibronectin and laminin in Smad2 activation in vascular endothelial cells in response to disturbed flow

BackgroundAtherosclerosis occurs in arterial curvatures and branches, where the flow is disturbed with low and oscillatory shear stress (OSS). The remodeling and alterations of extracellular matrices (ECMs) and their composition is the critical step in atherogenesis. In this study, we investigated the effects of different ECM proteins on the regulation of mechanotransduction in vascular endothelial cells (ECs) in response to OSS.MethodsThrough the experiments ranging from in vitro cell culture studies on effects of OSS on molecular signaling to in vivo examinations on clinical specimens from patients with coronary artery disease (CAD), we elucidated the roles of integrins and different ECMs, i.e., fibronectin (FN) and laminin (LM), in transforming growth factor (TGF)-β receptor (TβR)-mediated Smad2 activation and nuclear factor-κB (NF-κB) signaling in ECs in response to OSS and hence atherogenesis.ResultsOSS at 0.5±12 dynes/cm2 induces sustained increases in the association of types I and II TβRs with β1 and β3 integrins in ECs grown on FN, but it only transient increases in ECs grown on LM. OSS induces a sustained activation of Smad2 in ECs on FN, but only a transient activation of Smad2 in ECs on LM. OSS-activation of Smad2 in ECs on FN regulates downstream NF-κB signaling and pro-inflammatory gene expression through the activation of β1 integrin and its association with TβRs. In contrast, OSS induces transient activations of β1 and β3 integrins in ECs on LM, which associate with type I TβR to regulate Smad2 phosphorylation, resulting in transient induction of NF-κB and pro-inflammatory gene expression. In vivo investigations on diseased human coronary arteries from CAD patients revealed that Smad2 is highly activated in ECs of atherosclerotic lesions, which is accompanied by the concomitant increase of FN rather than LM in the EC layer and neointimal region of atherosclerotic lesions.ConclusionsOur findings provide new insights into the mechanisms of how OSS regulates Smad2 signaling and pro-inflammatory genes through the complex signaling networks of integrins, TβRs, and ECMs, thus illustrating the molecular basis of regional pro-inflammatory activation within disturbed flow regions in the arterial tree.

Protein kinase C-δ and -β coordinate flow-induced directionality and deformation of migratory human blood T-lymphocytes

T-lymphocyte migration under flow is critical for immune responses, but the mechanisms by which flow modulates the migratory behaviors of T-lymphocytes remain unclear. Human peripheral blood T-lymphocytes (PBTLs), when stimulated with phorbol 12-myristate 13-acetate (PMA), stretched their cell bodies dramatically and moved along the flow direction. In contrast, stromal cell-derived factor-1α-stimulated PBTLs deformed and migrated in a random manner. Here we elucidated the molecular mechanisms underlying flow-induced directionality and deformation of PMA-stimulated PBTLs. PMA primed PBTLs for polarization under flow, with protein kinase C (PKC)-δ enriched in the leading edge, PKC-βI in the microtubule organizing center, and PKC-βII in the uropod and peripheral region. PKC-δ regulated cell protrusions in the leading edge through Tiam1/Rac1/calmodulin, whereas PKC-β regulated RhoA/Rho-associated kinase activity and microtubule stability to modulate uropod contractility and detachment. Our findings indicate that PKC-δ and -β coordinate in the cell leading edge and uropod, respectively, to modulate the directionality and deformability of migratory T-lymphocytes under flow.