Regulation of PD-L1 expression by matrix stiffness in lung cancer cells

Expression of programmed death-ligand 1 (PD-L1) in tumor cells such as lung cancer cells plays an important role in mechanisms underlying evasion of an immune check point system. Lung cancer tissue with increased deposition of extracellular matrix is much stiffer than normal lung tissue. There is emerging evidence that the matrix stiffness of cancer tissue affects the phenotypes and properties of cancer cells. Nevertheless, the effects of substrate rigidity on expression of PD-L1 in lung cancer cells remain elusive. We evaluated the effects of substrate stiffness on PD-L1 expression in HCC827 lung adenocarcinoma cells by using polyacrylamide hydrogels with stiffnesses of 2 and 25?kPa. Expression of PD-L1 protein was higher on the stiffer substrates (25?kPa gel and plastic dish) than on the soft 2?kPa gel. PD-L1 expression was reduced by detachment of cells adhering to the substrate. Interferon-γ enhanced expression of PD-L1 protein cultured on stiff (25?kPa gel and plastic dishes) and soft (2?kPa gel) substrates and in the cell adhesion-free condition. As the stiffness of substrates increased, formation of actin stress fiber and cell growth were enhanced. Transfection of the cells with short interfering RNA for PD-L1 inhibited cell growth without affecting stress fiber formation. Treatment of the cells with cytochalasin D, an inhibitor of actin polymerization, significantly reduced PD-L1 protein levels. Taken together, a stiff substrate enhanced PD-L1 expression via actin-dependent mechanisms in lung cancer cells. It is suggested that stiffness as a tumor environment regulates PD-L1 expression, which leads to evasion of the immune system and tumor growth.     

Functions and Regulation of Circular Dorsal Ruffles

Cells construct a number of plasma membrane structures to meet a range of physiological demands. Driven by juxtamembrane actin machinery, these actin-based membrane protrusions are essential for the operation and maintenance of cellular life. They are required for diverse cellular functions, such as directed cell motility, cell spreading, adhesion, and substrate/matrix degradation. Circular dorsal ruffles (CDRs) are one class of such structures characterized as F-actin-rich membrane projections on the apical cell surface. CDRs commence their formation minutes after stimulation as flat, open, and immature ruffles and progressively develop into fully enclosed circular ruffles. These “rings” then mature and contract centrifugally before subsiding. Serving a critical function in receptor internalization and cell migration, CDRs are thus highly dynamic but transient formations. Here, we review the current state of knowledge concerning the regulation of circular dorsal ruffles. We focus specifically on the biochemical pathways leading to CDR formation in order to better define the roles and functions of these enigmatic structures.     

E3 ubiquitin ligases in regulating stress fiber,lamellipodium, and focal adhesion dynamics

Recent discoveries have unveiled the roles of a complicated network of E3 ubiquitin ligases in regulating cell migration machineries. The E3 ubiquitin ligases Smurf1 and Cul/BACURD ubiquitinate RhoA to regulate stress fiber formation and cell polarity, and ASB2α ubiquitinates filamins to modulate cytoskeletal stiffness, thus regulating cell spreading and cell migration. HACE1, XIAP, and Skp1-Cul1-F-box bind to Rac1 and cause its ubiquitination and degradation, thus suppressing lamellipodium protrusions, while PIAS3, a SUMO ligase, activates Rac1 to promote lamellipodium dynamics. Smurf1 also enhances Rac1 activation but it does not ubiquitinate Rac1. Both Smurf1 and HECTD1 regulate focal adhesion (FA) assembly and (or) disassembly through ubiquitinating the talin head domain and phosphatidylinositol 4 phosphate 5-kinase type I γ (PIPKIγ90), respectively. Thus, E3 ubiquitin ligases regulate stress fiber formation, cell polarity, lamellipodium protrusions, and FA dynamics through ubiquitinating the key proteins that control these processes.     

Elasticity Maps of Living Neurons Measured by Combined Fluorescence and Atomic Force Microscopy

Detailed knowledge of mechanical parameters such as cell elasticity, stiffness of the growth substrate, or traction stresses generated during axonal extensions is essential for understanding the mechanisms that control neuronal growth. Here, we combine atomic force microscopy-based force spectroscopy with fluorescence microscopy to produce systematic, high-resolution elasticity maps for three different types of live neuronal cells: cortical (embryonic rat), embryonic chick dorsal root ganglion, and P-19 (mouse embryonic carcinoma stem cells) neurons. We measure how the stiffness of neurons changes both during neurite outgrowth and upon disruption of microtubules of the cell. We find reversible local stiffening of the cell during growth, and show that the increase in local elastic modulus is primarily due to the formation of microtubules. We also report that cortical and P-19 neurons have similar elasticity maps, with elastic moduli in the range 0.1–2 kPa, with typical average values of 0.4 kPa (P-19) and 0.2 kPa (cortical). In contrast, dorsal root ganglion neurons are stiffer than P-19 and cortical cells, yielding elastic moduli in the range 0.1–8 kPa, with typical average values of 0.9 kPa. Finally, we report no measurable influence of substrate protein coating on cell body elasticity for the three types of neurons.     

Computational model predicts cell orientation in response to a range of mechanical stimuli

To build anisotropic, mechanically functioning tissue, it is essential to understand how cells orient in response to mechanical stimuli. Therefore, a computational model was developed which predicts cell orientation, based on the actin stress fiber distribution inside the cell. In the model, the stress fiber distribution evolves dynamically according to the following: (1) Stress fibers contain polymerized actin. The total amount of depolymerized plus polymerized actin is constant. (2) Stress fibers apply tension to their environment. This active tension is maximal when strain rate and absolute strain are zero and reduces with increasing shortening rate and absolute strain. (3) A high active fiber stress in a direction leads to a large amount of fibers in this direction. (4) The cell is attached to a substrate; all fiber stresses are homogenized into a total cell stress, which is in equilibrium with substrate stress. This model predicts that on a substrate of anisotropic stiffness, fibers align in the stiffest direction. Under cyclic strain when the cellular environment is so stiff that no compaction occurs (1 MPa), the model predicts strain avoidance, which is more pronounced with increasing strain frequency or amplitude. Under cyclic strain when the cellular environment is so soft that cells can compact it (10 kPa), the model predicts a preference for the cyclically strained compared to the compacting direction. These model predictions all agree with experimental evidence. For the first time, a computational model predicts cell orientation in response to this range of mechanical stimuli using a single set of parameters.     

p53 Down Regulates PDGF-Induced Formation of Circular Dorsal Ruffles in Rat Aortic Smooth Muscle Cells

The tumor suppressor, p53, negatively regulates cell migration and invasion in addition to its role in apoptosis, cell cycle regulation and senescence. Here, we study the roles of p53 in PDGF-induced circular dorsal ruffle (CDR) formation in rat aortic smooth muscle (RASM) cells. In primary and immortalized RASM cells, up-regulation of p53 expression or increase in activity with doxorubicin inhibits CDR formation. In contrast, shRNA-knockdown of p53 or inhibition of its activity with pifithrin α promotes CDR formation. p53 acts by up-regulating PTEN expression, which antagonizes Rac and Cdc42 activation. Both lipid and protein phosphatase activities of PTEN are required for maximal suppression of CDR, but the lipid activity clearly plays the dominant role. N-WASP, the downstream effector of Cdc42, is the major positive contributor to CDR formation in RASM, and is an indirect target of p53. The Rac effector, WAVE2, appears to also play a minor role, while WAVE1 has no significant effect in CDR formation. In sum, we propose that p53 suppresses PDGF-induced CDR formation in RASM cells by upregulating PTEN leading mainly to the inhibition of the Cdc42-N-WASP pathway.     

ADP-ribosylation factor 6 regulates actin cytoskeleton remodeling in coordination with Rac1 and RhoA

In this study, we have documented an essential role for ADP-ribosylation factor 6 (ARF6) in cell surface remodeling in response to physiological stimulus and in the down regulation of stress fiber formation. We demonstrate that the G-protein-coupled receptor agonist bombesin triggers the redistribution of ARF6- and Rac1-containing endosomal vesicles to the cell surface. This membrane redistribution was accompanied by cortical actin rearrangements and was inhibited by dominant negative ARF6, implying that bombesin is a physiological trigger of ARF6 activation. Furthermore, these studies provide a new model for bombesin-induced Rac1 activation that involves ARF6-regulated endosomal recycling. The bombesin-elicited translocation of vesicular ARF6 was mimicked by activated Galphaq and was partially inhibited by expression of RGS2, which down regulates Gq function. This suggests that Gq functions as an upstream regulator of ARF6 activation. The ARF6-induced peripheral cytoskeletal rearrangements were accompanied by a depletion of stress fibers. Moreover, cells expressing activated ARF6 resisted the formation of stress fibers induced by lysophosphatidic acid. We show that the ARF6-dependent inhibition of stress fiber formation was due to an inhibition of RhoA activation and was overcome by expression of a constitutively active RhoA mutant. The latter observations demonstrate that activation of ARF6 down regulates Rho signaling. Our findings underscore the potential roles of ARF6, Rac1, and RhoA in the coordinated regulation of cytoskeletal remodeling.     

Role of Actin Polymerization and Adhesion to Extracellular Matrix in Rac- and Rho-induced Cytoskeletal Reorganization

Most animal cells use a combination of actin-myosin–based contraction and actin polymerization– based protrusion to control their shape and motility. The small GTPase Rho triggers the formation of contractile stress fibers and focal adhesion complexes (Ridley, A.J., and A. Hall. 1992. Cell. 70:389–399) while a close relative, Rac, induces lamellipodial protrusions and focal complexes in the lamellipodium (Nobes, C.D., and A. Hall. 1995. Cell. 81:53–62; Ridley, A.J., H.F. Paterson, C.L. Johnston, D. Diekmann, and A. Hall. 1992. Cell. 70:401–410); the Rho family of small GTPases may thus play an important role in regulating cell movement. Here we explore the roles of actin polymerization and extracellular matrix in Rho- and Rac-stimulated cytoskeletal changes. To examine the underlying mechanisms through which these GTPases control F-actin assembly, fluorescently labeled monomeric actin, Cy3-actin, was introduced into serum-starved Swiss 3T3 fibroblasts. Incorporation of Cy3- actin into lamellipodial protrusions is concomitant with F-actin assembly after activation of Rac, but Cy3-actin is not incorporated into stress fibers formed immediately after Rho activation. We conclude that Rac induces rapid actin polymerization in ruffles near the plasma membrane, whereas Rho induces stress fiber assembly primarily by the bundling of actin filaments. Activation of Rho or Rac also leads to the formation of integrin adhesion complexes. Integrin clustering is not required for the Rho-induced assembly of actin-myosin filament bundles, or for vinculin association with actin bundles, but is required for stress fiber formation. Integrin-dependent focal complex assembly is not required for the Rac-induced formation of lamellipodia or membrane ruffles. It appears, therefore, that the assembly of large integrin complexes is not required for most of the actin reorganization or cell morphology changes induced by Rac or Rho activation in Swiss 3T3 fibroblasts.     

Rho- and Rac-dependent Assembly of Focal Adhesion Complexes and Actin Filaments in Permeabilized Fibroblasts: An Essential Role for Ezrin/Radixin/Moesin Proteins

The small GTPases Rho and Rac regulate actin filament assembly and the formation of integrin adhesion complexes to produce stress fibers and lamellipodia, respectively, in mammalian cells. Although numerous candidate effectors that might mediate these responses have been identified using the yeast two-hybrid and affinity purification techniques, their cellular roles remain unclear. We now describe a biological assay that allows components of the Rho and Rac signaling pathways to be identified. Permeabilization of serum-starved Swiss 3T3 cells with digitonin in the presence of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) induces both actin filament and focal adhesion complex assembly through activation of endogenous Rho and Rac. These responses are lost when GTPγS is added 6 min after permeabilization, but can be reconstituted using concentrated cytosolic extracts. We have achieved a 10,000-fold purification of the activity present in pig brain cytosol and protein sequence analysis shows it to contain moesin. Using recombinant proteins, we show that moesin and its close relatives ezrin and radixin can reconstitute stress fiber assembly, cortical actin polymerization and focal complex formation in response to activation of Rho and Rac.     

Role of myosin regulatory light chain and Rac1 in the migration of polyamine-depleted intestinal epithelial cells

We have previously shown that polyamine depletion decreased migration, Rac activation, and protein serine threonine phosphatase 2A activity. We have also shown that polyamine depletion increased cortical F-actin and decreased lamellipodia and stress fibers. In this study, we used staurosporine (STS), a potent, cell-permeable, and broad-spectrum serine/threonine kinase inhibitor, and studied migration. STS concentrations above 100 nM induced apoptosis. However, in polyamine-depleted cells, a lower concentration of STS (5 nM) increased attachment, spreading, Rac1 activation, and, subsequently, migration without causing apoptosis. STS-induced migration was completely prevented by a Rac1 inhibitor (NSC-23766) and dominant negative Rac1. These results imply that STS restores migration in polyamine-depleted cells through Rac1. The most important finding in this study was that polyamine depletion increased the association of phosphorylated myosin regulatory light chain (pThr(18)/Ser(19)-MRLC) at the cell periphery, which colocalized with thick cortical F-actin. Localization of pThr(18)- and pSer(19)-MRLC was found with stress fibers and nuclei, respectively. STS decreased the phosphorylation of cellular and peripheral pThr(18)-MRLC without any effect on nuclear pSer(19)-MRLC, dissolved thick cortical F-actin, and increased lamellipodia and stress fiber formation in polyamine-depleted cells. In control and polyamine-depleted cells, focal adhesion kinase (FAK) colocalized with stress fibers and the actin cortex, respectively. STS reorganized FAK, paxillin, and the cytoskeleton. These results suggest that polyamine depletion prevents the dephosphorylation of MRLC and thereby prevents the dynamic reorganization of the actin cytoskeleton and decreases lamellipodia formation resulting in the inhibition of migration.     

MEK1 restores migration of polyamine-depleted cells by retention and activation of Rac1 in the cytoplasm

We previously showed that polyamines are required for proliferation and migration both in vivo and in a cultured intestinal epithelial cell (IEC-6) model. Wounding of the IEC-6 monolayer induced transient ERK activation, which was further enhanced by EGF. EGF stimulated migration in control and polyamine-depleted cells, but the degree of stimulation was significantly less in polyamine-depleted cells. Inhibition of MEK1 inhibited basal as well as EGF-induced ERK activation and migration. Expression of constitutively active (CA)-MEK and dominant-negative (DN)-MEK had significant effects on F-actin structure. CA-MEK increased stress fiber and lamellipodia formation, while DN-MEK showed loss of stress fibers and abnormal actin cytoskeletal structure. Unlike EGF, CA-MEK significantly increased migration of both control and polyamine-depleted cells. The most important and significant finding in this study was that polyamine depletion caused localization of Rac1 and RhoA to the nuclear as well as perinuclear regions. Interestingly, CA-MEK completely reversed the subcellular distribution of Rac1 and RhoA proteins in polyamine-depleted cells. Polyamine depletion increased Rac1 in the nuclear fraction and decreased it in the cytoplasmic and membrane fractions of vector-transfected cells. CA-MEK prevented accumulation of Rac1 in the nucleus. Polyamine depletion significantly decreased Rac1 activity during 6-h migration in vector-transfected cells. Cells transfected with CA-MEK had almost identical levels of activated Rac1 in all three groups. These results suggest that polyamine depletion prevents activation of Rac1 and RhoA by sequestering them to the nucleus and that expression of constitutively active MEK reverses this effect, creating the cellular localization required for activation. epidermal growth factor; extracellular signal-regulated kinase; IEC-6 cells     

Effects of rho kinase and actin stress fibers on sustained extracellular signal-regulated kinase activity and activation of G(1) phase cyclin-dependent kinases

We recently reported that Rho kinase is required for sustained ERK signaling and the consequent mid-G(1) phase induction of cyclin D1 in fibroblasts. The results presented here indicate that these Rho kinase effects are mediated by the formation of stress fibers and the consequent clustering of alpha5beta1 integrin. Mechanistically, alpha5beta1 signaling and stress fiber formation allowed for the sustained activation of MEK, and this effect was mediated upstream of Ras-GTP loading. Interestingly, disruption of stress fibers with ML-7 led to G(1) phase arrest while comparable disruption of stress fibers with Y27632 (an inhibitor of Rho kinase) or dominant-negative Rho kinase led to a more rapid progression through G(1) phase. Inhibition of either MLCK or Rho kinase blocked sustained ERK signaling, but only Rho kinase inhibition allowed for the induction of cyclin D1 and activation of cdk4 via Rac/Cdc42. The levels of cyclin E, cdk2, and their major inhibitors, p21(cip1) and p27(kip1), were not affected by inhibition of MLCK or Rho kinase. Overall, our results indicate that Rho kinase-dependent stress fiber formation is required for sustained activation of the MEK/ERK pathway and the mid-G(1) phase induction of cyclin D1, but not for other aspects of cdk4 or cdk2 activation. They also emphasize that G(1) phase cell cycle progression in fibroblasts does not require stress fibers if Rac/Cdc42 signaling is allowed to induce cyclin D1.     

Sensitivity of alveolar macrophages to substrate mechanical and adhesive properties

In order to understand the sensitivity of alveolar macrophages (AMs) to substrate properties, we have developed a new model of macrophages cultured on substrates of increasing Young's modulus: (i) a monolayer of alveolar epithelial cells representing the supple (approximately 0.1 kPa) physiological substrate, (ii) polyacrylamide gels with two concentrations of bis-acrylamide representing low and high intermediate stiffness (respectively 40 kPa and 160 kPa) and, (iii) a highly rigid surface of plastic or glass (respectively 3 MPa and 70 MPa), the two latter being or not functionalized with type I-collagen. The macrophage response was studied through their shape (characterized by 3D-reconstructions of F-actin structure) and their cytoskeletal stiffness (estimated by transient twisting of magnetic RGD-coated beads and corrected for actual bead immersion). Macrophage shape dramatically changed from rounded to flattened as substrate stiffness increased from soft ((i) and (ii)) to rigid (iii) substrates, indicating a net sensitivity of alveolar macrophages to substrate stiffness but without generating F-actin stress fibers. Macrophage stiffness was also increased by large substrate stiffness increase but this increase was not due to an increase in internal tension assessed by the negligible effect of a F-actin depolymerizing drug (cytochalasine D) on bead twisting. The mechanical sensitivity of AMs could be partly explained by an idealized numerical model describing how low cell height enhances the substrate-stiffness-dependence of the apparent (measured) AM stiffness. Altogether, these results suggest that macrophages are able to probe their physical environment but the mechanosensitive mechanism behind appears quite different from tissue cells, since it occurs at no significant cell-scale prestress, shape changes through minimal actin remodeling and finally an AMs stiffness not affected by the loss in F-actin integrity.     

Paxillin mediates sensing of physical cues and regulates directional cell motility by controlling lamellipodia positioning

Physical interactions between cells and the extracellular matrix (ECM) guide directional migration by spatially controlling where cells form focal adhesions (FAs), which in turn regulate the extension of motile processes. Here we show that physical control of directional migration requires the FA scaffold protein paxillin. Using single-cell sized ECM islands to constrain cell shape, we found that fibroblasts cultured on square islands preferentially activated Rac and extended lamellipodia from corner, rather than side regions after 30 min stimulation with PDGF, but that cells lacking paxillin failed to restrict Rac activity to corners and formed small lamellipodia along their entire peripheries. This spatial preference was preceded by non-spatially constrained formation of both dorsal and lateral membrane ruffles from 5-10 min. Expression of paxillin N-terminal (paxN) or C-terminal (paxC) truncation mutants produced opposite, but complementary, effects on lamellipodia formation. Surprisingly, pax-/- and paxN cells also formed more circular dorsal ruffles (CDRs) than pax+ cells, while paxC cells formed fewer CDRs and extended larger lamellipodia even in the absence of PDGF. In a two-dimensional (2D) wound assay, pax-/- cells migrated at similar speeds to controls but lost directional persistence. Directional motility was rescued by expressing full-length paxillin or the N-terminus alone, but paxN cells migrated more slowly. In contrast, pax-/- and paxN cells exhibited increased migration in a three-dimensional (3D) invasion assay, with paxN cells invading Matrigel even in the absence of PDGF. These studies indicate that paxillin integrates physical and chemical motility signals by spatially constraining where cells will form motile processes, and thereby regulates directional migration both in 2D and 3D. These findings also suggest that CDRs may correspond to invasive protrusions that drive cell migration through 3D extracellular matrices.     

The Ig VH complementarity-determining region 3-containing Rb9 peptide,inhibits melanoma cells migration and invasion by interactions with Hsp90 and an adhesion G-protein coupled receptor

The present work aims at investigating the mechanism of action of the Rb9 peptide, which contains the V_HCDR 3 sequence of anti-sodium-dependent phosphate transport protein 2B (NaPi2B) monoclonal antibody RebMab200 and displayed antitumor properties. Short peptides corresponding to the hypervariable complementarity-determining regions (CDRs) of immunoglobulins have been associated with antimicrobial, antiviral, immunomodulatory and antitumor activities regardless of the specificity of the antibody. We have shown that the CDR derived peptide Rb9 induced substrate hyperadherence, inhibition of cell migration and matrix invasion in melanoma and other tumor cell lines. Rb9 also inhibited metastasis of murine melanoma in a syngeneic mouse model. We found that Rb9 binds to and interferes with Hsp90 chaperone activity causing attenuation of FAK-Src signaling and downregulation of active Rac1 in B16F10-Nex2 melanoma cells. The peptide also bound to an adhesion G-protein coupled receptor, triggering a concentration-dependent synthesis of cAMP and activation of PKA and VASP signaling as well as IP-3 dependent Ca²⁺ release. Hsp90 is highly expressed on the cell surface of melanoma cells, and synthetic agents that target Hsp90 are promising cancer therapeutic drugs. Based on their remarkable antitumor effects, the CDR-H3-derived peptides from RebMab200, and particularly the highly soluble and stable Rb9, are novel candidates to be further studied as potential antitumor drugs, selectively acting on cancer cell motility and invasion.     

Substrate stiffness affects the functional maturation of neonatal rat ventricular myocytes

Cardiac cells mature in the first postnatal week, concurrent with altered extracellular mechanical properties. To investigate the effects of extracellular stiffness on cardiomyocyte maturation, we plated neonatal rat ventricular myocytes for 7 days on collagen-coated polyacrylamide gels with varying elastic moduli. Cells on 10 kPa substrates developed aligned sarcomeres, whereas cells on stiffer substrates had unaligned sarcomeres and stress fibers, which are not observed in vivo. We found that cells generated greater mechanical force on gels with stiffness similar to the native myocardium, 10 kPa, than on stiffer or softer substrates. Cardiomyocytes on 10 kPa gels also had the largest calcium transients, sarcoplasmic calcium stores, and sarcoplasmic/endoplasmic reticular calcium ATPase2a expression, but no difference in contractile protein. We hypothesized that inhibition of stress fiber formation might allow myocyte maturation on stiffer substrates. Treatment of maturing cardiomyocytes with hydroxyfasudil, an inhibitor of RhoA kinase and stress fiber-formation, resulted in enhanced force generation on the stiffest gels. We conclude that extracellular stiffness near that of native myocardium significantly enhances neonatal rat ventricular myocytes maturation. Deviations from ideal stiffness result in lower expression of sarcoplasmic/endoplasmic reticular calcium ATPase, less stored calcium, smaller calcium transients, and lower force. On very stiff substrates, this adaptation seems to involve RhoA kinase.     

Rac1/MAPK/ERK 通路介导脂多糖LPS 诱导的人内皮细胞 单层通透性增高机制研究

目的:探讨LPS诱导的人内皮细胞单层通透性改变的分子机制。方法:应用逆转录病毒为载体,感染并筛选稳定表达持续活化型Rac1和主导抑制型Rac1的人HUVEC细胞,应用LPS刺激并观察细胞骨架蛋白F-actin和HUVEC单层通透性的改变。同时应用Western blot方法检测LPS刺激前后细胞中MAPK/ERK信号通路的改变及加入PD98059阻断ERK表达后,细胞内F-actin的改变情况。结果:与正常HUVEC相比较,LPS刺激后,感染活化型Rac1和主导抑制型Rac1的HUVEC中F-actin重构并形成大量应力纤维,细胞单层通透性显著增加。而抑制型Rac1感染后的HUVEC中F-actin无重构现象,同时细胞单层通透性无明显增加。LPS刺激前后,各组细胞中ERK1/2总蛋白均无明显改变。LPS刺激后,感染活化型Rac1的HUVEC中,p-ERK增加。经PD98059阻断后,细胞内p-ERK表达下降同时伴随F-actin解聚发生。结论:LPS诱导的内皮细胞通透性增加是经过Rac1-MAPK/ERK通路介导的。     

从抗TNF抗体的CDR肽库中获得拮抗TNF的短肽

为设计来自抗体的短肽 ,以抗肿瘤坏死因子 (TNF)嵌合抗体 (cA2 )CDRs为模板 ,在其两侧各加 3个随机氨基酸残基 ( X3 CDR X3 ) ,构建了 6个以CDR为基础的肽库 .经过 3轮亲和选择 ,挑取单克隆 ,进一步经ELISA检测TNF阳性噬菌体克隆 ,分离得到 7个ELISA阳性较好的噬菌体肽克隆 ,分别命名为CDR2L1、CDR2L2、CDR2L3、CDR1L1、CDR2H1、CDR3H1、CDR3H 2 .应用MTT方法 ,检测 7个克隆对TNF生物学活性的拮抗作用 .结果显示 :来自CDR2L ,CDR3H肽库中的CDR2L2、CDR2L3,CDR3H2噬菌体肽具有明显的拮抗TNF诱导L92 9细胞的细胞毒作用 ,其中以CDR2L2噬菌体肽的拮抗活性最强 .而来源于CDR1L ,CDR2H肽库的CDR1L1和CDR2H1噬菌体肽和来自CDR2L ,CDR3H肽库中的CDR2L1和CDR3H1噬菌体肽没有明显的拮抗TNF作用 .研究结果初步表明 :从cA2抗体CDR肽库中筛选得到的噬菌体CDR模拟肽具有亲本抗体相似的结合活性和生物学效应 ,从而为开发已知抗体 (特别是治疗用抗体 )CDR为基础的肽药物创建一个技术平台奠定基础     

Reorganization of actin cytoskeleton by the phosphoinositide metabolite glycerophosphoinositol 4-phosphate

Glycerophosphoinositol 4-phosphate (GroPIns-4P) is a biologically active, water-soluble phospholipase A metabolite derived from phosphatidylinositol 4-phosphate, whose cellular concentrations have been reported to increase in Ras-transformed cells. It is therefore important to understand its biological activities. Herein, we have examined whether GroPIns-4P can regulate the organization of the actin cytoskeleton, because this could be a Ras-related function involved in cell motility and metastatic invasion. We find that in serum-starved Swiss 3T3 cells, exogenously added GroPIns-4P rapidly and potently induces the formation of membrane ruffles, and, later, the formation of stress fibers. These actin structures can be regulated by the small GTPases Cdc42, Rac, and Rho. To analyze the mechanism of action of GroPIns-4P, we selectively inactivated each of these GTPases. GroPIns-4P requires active Rac and Rho, but not Cdc42, for ruffle and stress fiber formation, respectively. Moreover, GroPIns-4P induces a rapid translocation of the green fluorescent protein-tagged Rac into ruffles, and increases the fraction of GTP-bound Rac, in intact cells. The activation of Rac by GroPIns-4P was near maximal and long-lasting. Interestingly, this feature seems to be critical in the induction of actin ruffles by GroPIns-4P.     

Src, PKCα, and PKCδ are required for αvβ3 integrin-mediated metastatic melanoma invasion

Background

Integrins, cell-surface receptors that mediate adhesive interactions between cells and the extracellular matrix (ECM), play an important role in cancer progression. Expression of the vitronectin receptor αvβ3 integrin correlates with increased invasive and metastatic capacity of malignant melanomas, yet it remains unclear how expression of this integrin triggers melanoma invasion and metastasis.

Results

Two melanoma cell lines C8161.9 and M14 both express high levels of αvβ3 integrin and adhere to vitronectin. However, only the highly metastatic C8161.9 cells are capable of invading vitronectin-enriched Matrigel in an αvβ3-depenent manner. Elevated levels of PKCα and PKCδ, and activated Src were detected specifically in the highly metastatic melanoma cells, but not in the low metastatic M14 cells. Inhibition of Src or PKC activity suppressed αvβ3-dependent invasion. Furthermore, over expression of Src or PKCα and PKCδ was sufficient to confer αvβ3-dependent invasiveness to M14 cells. Stress fiber formation and focal adhesion formation were almost completely absent in C8161.9 cells compared to M14 cells. Inhibition of Src signaling was sufficient to restore normal actin architecture, and resulted in decreased p190RhoGAP phosphorylation and enhanced RhoA activity. Src had no effect on Rac activity. Loss of PKCα expression, but not PKCδ, by siRNA inhibited Rac and PAK activity as well as invasiveness. Loss of PKCα restored focal adhesion formation and partially restored stress fiber formation, while loss of PKCδ primarily restored stress fibers.

Conclusion

The misregulated expression of PKCα and PKCδ and elevated Src activity in metastatic melanoma cells is required for efficient αvβ3-mediated invasion. PKCα and Src enhance αvβ3-mediated invasion in part by increasing the GTPase activity of Rac relative to RhoA. PKCα influences focal adhesion formation, while PKCδ controls stress fibers.