The impact of glutaredoxin 1 and glutaredoxin 2 double knockout on lens epithelial cell function.

Glutaredoxins (Grx1 and Grx2) are thiol-repair antioxidant enzymes that play vital roles in cellular redox homeostasis and various cellular processes. This study aims to evaluate the functions of the glutaredoxin (Grx) system, including glutaredoxin 1 (Grx1) and glutaredoxin 2 (Grx2), using Grx1/Grx2 double knockout (DKO) mice as a model. We isolated primary lens epithelial cells (LECs) from wild-type (WT) and DKO mice for a series of in vitro analyses. Our results revealed that Grx1/Grx2 DKO LECs exhibited slower growth rates, reduced proliferation, and aberrant cell cycle distribution compared to WT cells. Elevated levels of ß-galactosidase activity were observed in DKO cells, along with a lack of caspase 3 activation, suggesting that these cells may be undergoing senescence. Additionally, DKO LECs displayed compromised mitochondrial function, characterized by decreased ATP production, reduced expression levels of oxidative phosphorylation (OXPHOS) complexes III and IV, and increased proton leak. A compensatory metabolic shift towards glycolysis was observed in DKO cells, indicating an adaptive response to Grx1/Grx2 deficiency. Furthermore, loss of Grx1/Grx2 affected cellular structure, leading to increased polymerized tubulin, stress fiber formation, and vimentin expression in LECs. In conclusion, our study demonstrates that Grx1/Grx2 double deletion in LECs results in impaired cell proliferation, aberrant cell cycle progression, disrupted apoptosis, compromised mitochondrial function, and altered cytoskeletal organization. These findings underscore the importance of Grx1 and Grx2 in maintaining cellular redox homeostasis and the consequences of their deficiency on cellular structure and function. Further research is needed to elucidate the precise molecular mechanisms underlying these observations and to investigate potential therapeutic strategies targeting Grx1 and Grx2 for various physiological processes and oxidative-stress related diseases such as cataract.

Inhibiting mitochondrial fission rescues degeneration in hereditary spastic paraplegia neurons.

Hereditary spastic paraplegias are characterized by lower limb spasticity resulting from degeneration of long corticospinal axons. SPG11 is one of the most common autosomal recessive hereditary spastic paraplegias, and the SPG11 protein spatacsin forms a complex with the SPG15 protein spastizin and heterotetrameric AP5 adaptor protein complex, which includes the SPG48 protein AP5Z1. Using the integration-free episomal method, we established SPG11 patient-specific induced pluripotent stem cells (iPSCs) from patient fibroblasts. We differentiated SPG11 iPSCs, as well as SPG48 iPSCs previously established, into cortical projection neurons and examined protective effects by targeting mitochondrial dynamics using P110, a peptide that selectively inhibits mitochondrial fission GTPase Drp1. P110 treatment mitigates mitochondrial fragmentation, improves mitochondrial motility, and restores mitochondrial health and ATP levels in SPG11 and SPG48 neurons. Neurofilament aggregations are increased in SPG11 and SPG48 axons, and these are also suppressed by P110. Similarly, P110 mitigates neurofilament disruption in both SPG11 and SPG48 knockdown cortical projection neurons, confirming the contribution of hereditary spastic paraplegia gene deficiency to subsequent neurofilament and mitochondrial defects. Strikingly, neurofilament aggregations in SPG11 and SPG48 deficient neurons double stain with ubiquitin and autophagy related proteins, resembling the pathological hallmark observed in SPG11 autopsy brain sections. To confirm the cause-effect relationship between the SPG11 mutations and disease phenotypes, we knocked-in SPG11 disease mutations to human embryonic stem cells (hESCs) and differentiated these stem cells into cortical projection neurons. Reduced ATP levels and accumulated neurofilament aggregations along axons are observed, and both are mitigated by P110. Furthermore, rescue experiment with expression of wild-type SPG11 in cortical projection neurons derived from both SPG11 patient iPSCs and SPG11 disease mutation knock-in hESCs leads to rescue of mitochondrial dysfunction and neurofilament aggregations in these SPG11 neurons. Finally, in SPG11 and SPG48 long-term cultures, increased release of phosphoNF-H, a biomarker for nerve degeneration, is significantly reduced by inhibiting mitochondrial fission pharmacologically using P110 and genetically using Drp1 shRNA. Taken together, our results demonstrate that impaired mitochondrial dynamics underlie both cytoskeletal disorganization and axonal degeneration in SPG11 and SPG48 neurons, highlighting the importance of targeting these pathologies therapeutically.

Effects of titanium dioxide microparticles and nanoparticles on cytoskeletal organization, cell adhesion, migration, and proliferation in human gingival fibroblasts in the presence of lipopolysaccharide.

BACKGROUND AND OBJECTIVE: Titanium wear particles may participate in the etiology of peri-implantitis. However, the influence of titanium wear particles on biological behavior of human gingival fibroblasts (HGFs) in the presence of LPS is still not clear. The present study demonstrated the effects of titanium dioxide micro- and nanoparticles (TiO2  MPs and NPs) on HGF cell viability, cytoskeletal organization, adhesion, migration, and proliferation in vitro, and LPS was used to mimic the in vivo condition. METHODS: Primary HGFs were treated with TiO2 MPs (primary particle size <5 µm, 0.1 mg/ml) and NPs (primary particle size <100 nm, 0.1 mg/ml) with or without 1 µg/ml LPS. The effects of TiO2 MPs and NPs on HGFs cell viability was measured by CCK-8 assay. The proliferation of HGF was detected by Ki67 nuclear staining. The confocal laser scanning microscope (CLSM) was used to detect the internalization of TiO2 MPs and NPs in HGFs as well as the arrangement of F-actin, vinculin, and vimentin organization. Wound healing assay and transwell assay were performed to measure the migration of HGFs induced by TiO2 MPs and NPs. Cell adhesion was measured using fibronectin-coated plates. The relative mRNA and protein expression of adhesion relative protein such as focal adhesion kinase (FAK), fibronectin (FN), and type I collagen (COL1) were measured using quantitative RT-PCR and western blot analysis. One-way analysis of variance (ANOVA) and Student's t-test were used to analyze the statistical significance, and p < .05 was considered statistically significant. RESULTS: TiO2 NPs significantly inhibited HGF cell viability, proliferation, and migration compared with TiO2 MPs group and control group. Compared with control group (2.64 ± 0.09), the mean absorbance of the cells in 1 mg/ml TiO2 MPs group and 0.25 mg/ml TiO2 NPs group were significantly decreased to 1.93 ± 0.33 (p < .05) and 2.22 ± 0.18 (p < .01), respectively. The cytoskeleton disruption was found in TiO2 NPs group. The mRNA and protein expression were significantly downregulated by TiO2 NPs. Furthermore, both TiO2 NPs and MPs induced more adverse effects on HGFs in the presence of LPS. CONCLUSION: Our results indicate that TiO2 NPs but not TiO2 MPs significantly disrupt the cytoskeletal organization and inhibited cell adhesion, migration, and proliferation of HGFs. However, in the presence of LPS, TiO2 MPs, and TiO2 NPs enhance these negative effects in HGFs. Titanium wear particles are probably involved in the initiation and progression of peri-implant diseases.

Protein Kinase D1, Reduced in Human Pancreatic Tumors, Increases Secretion of Small Extracellular Vesicles From Cancer Cells That Promote Metastasis to Lung in Mice.

BACKGROUND & AIMS: Pancreatic tumor cells release small extracellular vesicles (sEVs, exosomes) that contain lipids and proteins, RNA, and DNA molecules that might promote formation of metastases. It is not clear what cargo these vesicles contain and how they are released. Protein kinase D1 (PRKD1) inhibits cell motility and is believed to be dysregulated in pancreatic ductal adenocarcinomas. We investigated whether it regulates production of sEVs in pancreatic cancer cells and their ability to form premetastatic niches for pancreatic cancer cells in mice. METHODS: We analyzed data from UALCAN and human pancreatic tissue microarrays to compare levels of PRKD1 between tumor and nontumor tissues. We studied mice with pancreas-specific disruption of Prkd1 (PRKD1KO mice), mice that express oncogenic KRAS (KC mice), and KC mice with disruption of Prkd1 (PRKD1KO-KC mice). Subcutaneous xenograft tumors were grown in NSG mice from Panc1 cells; some mice were then given injections of sEVs. Pancreata and lung tissues from mice were analyzed by histology, immunohistochemistry, and/or quantitative polymerase chain reaction; we performed nanoparticle tracking analysis of plasma sEVs. The Prkd1 gene was disrupted in Panc1 cells using CRISPR-Cas9 or knocked down with small hairpin RNAs, or PRKD1 activity was inhibited with the selective inhibitor CRT0066101. Pancreatic cancer cell lines were analyzed by gene-expression microarray, quantitative polymerase chain reaction, immunoblot, and immunofluorescence analyses. sEVs secreted by Panc1 cell lines were analyzed by flow cytometry, transmission electron microscopy, and mass spectrometry. RESULTS: Levels of PRKD1 were reduced in human pancreatic ductal adenocarcinoma tissues compared with nontumor tissues. PRKD1KO-KC mice developed more pancreatic intraepithelial neoplasia, at a faster rate, than KC mice, and had more lung metastases and significantly shorter average survival time. Serum from PRKD1KO-KC mice had increased levels of sEVs compared with KC mice. Pancreatic cancer cells with loss or inhibition of PRKD1 increased secretion of sEVs; loss of PRKD1 reduced phosphorylation of its substrate, cortactin, resulting in increased F-actin levels at the plasma membrane. sEVs from cells with loss or reduced expression of PRKD1 had altered content, and injection of these sEVs into mice increased metastasis of xenograft tumors to lung, compared with sEVs from pancreatic cells that expressed PRKD1. PRKD1-deficient pancreatic cancer cells showed increased loading of integrin α6ß4 into sEVs-a process that required CD82. CONCLUSIONS: Human pancreatic ductal adenocarcinoma has reduced levels of PRKD1 compared with nontumor pancreatic tissues. Loss of PRKD1 results in reduced phosphorylation of cortactin in pancreatic cancer cell lines, resulting in increased in F-actin at the plasma membrane and increased release of sEVs, with altered content. These sEVs promote metastasis of xenograft and pancreatic tumors to lung in mice.

Role of Periostin in Cardiac Valve Development.

Although periostin plays a significant role in adult cardiac remodeling diseases, the focus of this review is on periostin as a valvulogenic gene. Periostin is expressed throughout valvular development, initially being expressed in endocardial endothelial cells that have been activated to transform into prevalvular mesenchyme termed "cushion tissues" that sustain expression of periostin throughout their morphogenesis into mature (compacted) valve leaflets. The phenotype of periostin null indicates that periostin is not required for endocardial transformation nor the proliferation of its mesenchymal progeny but rather promotes cellular behaviors that promote migration, survival (anti-apoptotic), differentiation into fibroblastic lineages, collagen secretion and postnatal remodeling/maturation. These morphogenetic activities are promoted or coordinated by periostin signaling through integrin receptors activating downstream kinases in cushion cells that activate hyaluronan synthetase II (Akt/PI3K), collagen synthesis (Erk/MapK) and changes in cytoskeletal organization (Pak1) which regulate postnatal remodeling of cells and associated collagenous matrix into a trilaminar (zonal) histoarchitecture. Pak1 binding to filamin A is proposed as one mechanism by which periostin supports remodeling. The failure to properly remodel cushions sets up a trajectory of degenerative (myxomatous-like) changes that over time reduce biomechanical properties and increase chances for prolapse, regurgitation or calcification of the leaflets. Included in the review are considerations of lineage diversity and the role of periostin as a determinant of mesenchymal cell fate.

Emerging roles of TRIO and F-actin-binding protein in human diseases.

TRIO and F-actin-binding protein (TRIOBP) also referred to as Tara, was originally isolated as a cytoskeleton remodeling protein. TRIOBP-1 is important for regulating F-actin filament reorganization. TRIOBP variants are broadly classified as variant-1 or - 4 and do not share exons. TRIOBP variant-5 contains all exons. Earlier studies indicated that TRIOBP-4/5 mutation is a pivotal element of autosomal recessive nonsyndromic hearing loss. However, recent studies provide clues that TRIOBP variants are associated with other human diseases including cancer and brain diseases. In this review, recent functional studies focusing on TRIOBP variants and its possible disease models are described.

Engineered Models of Confined Cell Migration.

Cells in the body are physically confined by neighboring cells, tissues, and the extracellular matrix. Although physical confinement modulates intracellular signaling and the underlying mechanisms of cell migration, it is difficult to study in vivo. Furthermore, traditional two-dimensional cell migration assays do not recapitulate the complex topographies found in the body. Therefore, a number of experimental in vitro models that confine and impose forces on cells in well-defined microenvironments have been engineered. We describe the design and use of microfluidic microchannel devices, grooved substrates, micropatterned lines, vertical confinement devices, patterned hydrogels, and micropipette aspiration assays for studying cell responses to confinement. Use of these devices has enabled the delineation of changes in cytoskeletal reorganization, cell-substrate adhesions, intracellular signaling, nuclear shape, and gene expression that result from physical confinement. These assays and the physiologically relevant signaling pathways that have been elucidated are beginning to have a translational and clinical impact.

Deficiencies in extrusion of the second polar body due to high calcium concentrations during in vitro fertilization in inbred C3H/He mice.

Successful in vitro fertilization (IVF) of all inbred strains of laboratory mice has not yet been accomplished. We have previously shown that a high calcium concentration improved IVF in various inbred mice. However, we also found that in cumulus-free ova of C3H/He mice such IVF conditions significantly increased the deficiency of extrusion of the second polar body (PBII) in a dose-dependent manner (2% at 1.71 mM and 29% at 6.84 mM, P < 0.05) and that PBII extrusion was affected by high calcium levels at 2-3 h post-insemination. While developmental competence of ova without PBII extrusion to blastocysts after 96 h culture was not affected, a significant reduction in the nuclear number of the inner cell mass was observed in blastocyst fertilized under high calcium condition. We also examined how high calcium concentration during IVF affects PBII extrusion in C3H/He mice. Cumulus cells cultured under high calcium conditions showed a significantly alleviated deficient PBII extrusion. This phenomenon is likely to be specific to C3H/He ova because deficient PBII extrusion in reciprocal fertilization between C3H and BDF1 gametes was observed only in C3H/He ova. Sperm factor(s) was still involved in deficient PBII extrusion due to high calcium concentrations, as this phenomenon was not observed in ova activated by ethanol. The cytoskeletal organization of ova without PBII extrusion showed disturbed spindle rotation, incomplete formation of contractile ring and disturbed localization of actin, suggesting that high calcium levels affect the anchoring machinery of the meiotic spindle. These results indicate that in C3H/He mice high calcium levels induce abnormal fertilization, i.e. deficient PBII extrusion by affecting the cytoskeletal organization, resulting in disturbed cytokinesis during the second meiotic division. Thus, use of high calcium media for IVF should be avoided for this strain.

Desmin Reorganization by Stimuli Inducing Oxidative Stress and Electrophiles: Role of Its Single Cysteine Residue.

The type III intermediate filament proteins vimentin and GFAP are modulated by oxidants and electrophiles, mainly through perturbation of their single cysteine residues. Desmin, the type III intermediate filament protein specific to muscle cells, is critical for muscle homeostasis, playing a key role in sarcomere organization and mitochondrial function. Here, we have studied the impact of oxidants and cysteine-reactive agents on desmin behavior. Our results show that several reactive species and drugs induce covalent modifications of desmin in vitro, of which its single cysteine residue, C333, is an important target. Moreover, stimuli eliciting oxidative stress or lipoxidation, including H2O2, 15-deoxy-prostaglandin J2, and CoCl2-elicited chemical hypoxia, provoke desmin disorganization in H9c2 rat cardiomyoblasts transfected with wild-type desmin, which is partially attenuated in cells expressing a C333S mutant. Notably, in cells lacking other cytoplasmic intermediate filaments, network formation by desmin C333S appears less efficient than that of desmin wt, especially when these proteins are expressed as fluorescent fusion constructs. Nevertheless, in these cells, the desmin C333S organization is also protected from disruption by oxidants. Taken together, our results indicate that desmin is a target for oxidative and electrophilic stress, which elicit desmin remodeling conditioned by the presence of its single cysteine residue.

Effect of GNE Mutations on Cytoskeletal Network Proteins: Potential Gateway to Understand Pathomechanism of GNEM.

GNE myopathy is an inherited neuromuscular disorder caused by mutations in GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase) gene catalyzing the sialic acid biosynthesis pathway. The characteristic features include muscle weakness in upper and lower extremities, skeletal muscle wasting, and rimmed vacuole formation. More than 200 GNE mutations in either epimerase or kinase domain have been reported worldwide. In Indian subcontinent, several GNE mutations have been recently identified with unknown functional correlation. Alternate role of GNE in various cellular processes such as cell adhesion, migration, apoptosis, protein aggregation, and cytoskeletal organization have been proposed in recent studies. We aim to understand and compare the effect of various GNE mutations from Indian origin on regulation of the cytoskeletal network. In particular, F-actin dynamics was determined quantitatively by determining F/G-actin ratios in immunoblots for specific proteins. The extent of F-actin polymerization was visualized by immunostaining with Phalloidin using confocal microscopy. The proteins regulating F-actin dynamics such as RhoA, cofilin, Arp2, and alpha-actinin were studied in various GNE mutants. The altered level of cytoskeletal organization network proteins affected cell migration of GNE mutant proteins as measured by wound healing assay. The functional comparison of GNE mutations will help in better understanding of the genotypic severity of the disease in the Indian population. Our study offers a potential for identification of therapeutic molecules regulating actin dynamics in GNE specific mutations.

ALD-R491 regulates vimentin filament stability and solubility, cell contractile force, cell migration speed and directionality.

Metastasizing cells express the intermediate filament protein vimentin, which is used to diagnose invasive tumors in the clinic. However, the role of vimentin in cell motility, and if the assembly of non-filamentous variants of vimentin into filaments regulates cell migration remains unclear. We observed that the vimentin-targeting drug ALD-R491 increased the stability of vimentin filaments, by reducing filament assembly and/or disassembly. ALD-R491-treatment also resulted in more bundled and disorganized filaments and an increased pool of non-filamentous vimentin. This was accompanied by a reduction in size of cell-matrix adhesions and increased cellular contractile forces. Moreover, during cell migration, cells showed erratic formation of lamellipodia at the cell periphery, loss of coordinated cell movement, reduced cell migration speed, directionality and an elongated cell shape with long thin extensions at the rear that often detached. Taken together, these results indicate that the stability of vimentin filaments and the soluble pool of vimentin regulate the speed and directionality of cell migration and the capacity of cells to migrate in a mechanically cohesive manner. These observations suggest that the stability of vimentin filaments governs the adhesive, physical and migratory properties of cells, and expands our understanding of vimentin functions in health and disease, including cancer metastasis.

Corrigendum: Overexpression Cathepsin D Contributes to Perineural Invasion of Salivary Adenoid Cystic Carcinoma.

[This corrects the article DOI: 10.3389/fonc.2018.00492.].

Rac1-GTPase regulates compression-induced actin protrusions (CAPs) of mesenchymal stem cells in 3D collagen micro-tissues.

Cells can sense mechanical signals through cytoskeleton reorganization. We previously discovered the formation of omni-directional actin protrusions upon compression loading, namely compression-induced actin protrusions (CAPs), in human mesenchymal stem cells (MSCs) in 3D micro-tissues. Here, the regulatory roles of three RhoGTPases (CDC42, Rac1 and RhoA) in the formation of CAPs were investigated. Upon compression loading, extensive formation of CAPs was found, significantly associated with an upregulated mRNA expression of Rac1 only, but not CDC42, nor RhoA. Upon chemical inhibition of these RhoGTPase activity during compression, only Rac1 activity was significantly suppressed, associating with the reduced CAP formation. Silencing the upstream regulators of these RhoGTPase pathways including Rac1 by specific siRNA dramatically disrupted actin cytoskeleton, distorted cell morphology and aborted CAP formation. Silencing cortactin (CTTN), a downstream effector of the Rac1 pathway, induced a compensatory upregulation of Rac1, enabling the MSCs to respond to the compression loading stimulus in terms of CAP formation, although at a reduced number. The importance of Rac1 signalling in CAP formation and the corresponding upregulation of lamellipodial markers also suggest that these CAPs are lamellipodia in nature. This study delineates the mechanism of compression-induced cytoskeleton reorganization, contributing to rationalizing mechanical loading regimes for functional tissue engineering.

Overexpression Cathepsin D Contributes to Perineural Invasion of Salivary Adenoid Cystic Carcinoma.

Objective: Cathepsin D (CTSD) is a pivotal orchestrator in the occurrence and development of tumors. Recently, CTSD was detected in salivary adenoid cystic carcinoma (SACC). However, its functional role in perineural invasion (PNI) of SACC remained elusive. We conducted the present study to detect the expression of CTSD in SACC, analyze the correlation between CTSD expression and prognosis of SACC patients and elucidate the role of CTSD in occurrence of PNI in SACC to lay the foundation for further studies. Methods: Immunohistochemical analysis was conducted to assess CTSD and Ki67 expression in 158 SACC samples and 20 normal salivary gland samples adjacent to carcinoma. Meanwhile, the correlation between CTSD and PNI of SACC specimens was analyzed using Wilcoxon test. QRT-PCR, immunofluorescence and western blot analysis were used to examine the levels of CTSD mRNA and protein in SACC-LM cell line. SiRNA-mediated CTSD silence was performed. Scratch wound healing assay, transwell invasion assay and DRG co-culture assay of PNI was used to detect the ability of migration, invasion and PNI. FITC-phalloidin was used to detect cytoskeletal organization. Results: Our data demonstrated that the positive expression of CTSD was observed in 74.1% (117/158) of SACC cases, and the expression of CTSD was significantly correlated with the PNI (p < 0.05). The ability of migration, invasion, and PNI could be inhibited significantly by siRNA-mediated CTSD silence (p < 0.01). Furthermore, siRNA-mediated CTSD silence inhibited cytoskeletal organization and pseudo foot formation in SACC-LM cells. Conclusion: Our results suggested that an association between PNI and expression of CTSD existed. CTSD may promote PNI of SACC accompanied by cytoskeletal organization and pseudo foot formation.

Hyaluronic Acid Dictates Chondrocyte Morphology and Migration in Composite Gels.

Tissue equivalent collagen-hyaluronic acid-based hydrogels are widely used for cartilage tissue engineering; however, not much importance has been given to investigate how cellular responses are altered with varying concentrations of hyaluronic acid in gels. In this study, different concentrations of hyaluronic acid dialdehyde (HAD) were combined with collagen to fabricate collagen-HAD composite (CH) gels, and the influence of HAD on cell shape, migration, viability, cytoskeletal organization, and gel contraction was examined. The microstructure and the mechanical strength of the composite gels were altered by varying HAD concentrations. Morphology of chondrocytes cultured on CH gels showed a significant increase in their aspect ratio and decrease in number of cell protrusions with increase in concentration of HAD. The organization of the cytoskeleton at the cellular protrusions was vimentin localized at the base, microtubules at the tip, and actin localized throughout the cell body. Changes in HAD concentrations altered hydrogel mechanical strength, cytoskeletal organization, and formation of cellular protrusions, all of which contributed to changes in cell morphology and migration. These changes were more evident in 3D cell-encapsulated gels than chondrocytes cultured over the 2D gels. However, viability of cells and matrix contraction, staining for adhesion protein vinculin, and hyaluronic acid receptor CD44 remained similar in all CH compositions. The changes in cell responses further influenced extracellular matrix deposition during in vitro culture. Cell responses in low HAD gels mimic the cellular behavior in damaged cartilage, whereas those in high HAD gels resembled the behavior in healthy cartilage tissue. Our study illustrates the importance of careful formulations of hydrogel compositions in designing biomimetic matrices that are used as in vitro models to study chondrocyte behavior.

Plant Endoplasmic Reticulum-Plasma Membrane Contact Sites.

The endoplasmic reticulum (ER) acts as a superhighway with multiple sideroads that connects the different membrane compartments including the ER to the plasma membrane (PM). ER-PM contact sites (EPCSs) are a common feature in eukaryotic organisms, but have not been studied well in plants owing to the lack of molecular markers and to the difficulty in resolving the EPCS structure using conventional microscopy. Recently, however, plant protein complexes required for linking the ER and PM have been identified. This is a further step towards understanding the structure and function of plant EPCSs. We highlight some recent studies in this field and suggest several hypotheses that relate to the possible function of EPCSs in plants.

In vitro reconstitution of dynamic microtubules interacting with actin filament networks.

Interactions between microtubules and actin filaments (F-actin) are essential for eukaryotic cell migration, polarization, growth, and division. Although the importance of these interactions has been long recognized, the inherent complexity of the cell interior hampers a detailed mechanistic study of how these two cytoskeletal systems influence each other. In this chapter, we show how in vitro reconstitution can be employed to study how actin filaments and dynamic microtubules affect each other's organization. While we focus here on the effect of steric interactions, these assays provide an ideal starting point to develop more complex studies through the addition of known F-actin-microtubule cross-linkers, or myosin II motors.

Inhibitory effects of obovatol on osteoclast differentiation and bone resorption.

Osteoclasts are polykaryons that have the unique capacity to degrade bone. Modulation of osteoclast formation and function is a promising strategy for the treatment of bone-destructive diseases. Here, we report that obovatol, a natural compound isolated from Magnolia obovata, inhibits receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL)-induced osteoclast differentiation in vitro and inflammatory bone loss in vivo. We found that obovatol strongly inhibited osteoclast formation from bone marrow-derived macrophages in a dose-dependent manner without cytotoxicity. Obovatol significantly suppressed RANKL-induced activation of NF-κB, c-Jun-N-terminal kinase, and extracellular signal-regulated kinase signaling pathways. Obovatol also inhibited RANKL-induced expression of the genes c-Fos and nuclear factor of activated T cells c1, which are transcription factors important for osteoclastogenesis. In addition to osteoclast differentiation, obovatol blocked cytoskeletal organization and abrogated the bone resorbing activity of mature osteoclast. Obovatol also accelerated osteoclast apoptosis through the induction of caspase-3 activation. Consistent with its in vitro anti-resorptive effect, obovatol prevented bone loss induced by lipopolysaccharide in vivo. Together, our data suggest that obovatol may be a useful therapeutic agent for the treatment of pathological bone disorders characterized by excessive osteoclastic bone resorption.

Fisetin antagonizes cell fusion, cytoskeletal organization and bone resorption in RANKL-differentiated murine macrophages.

Osteoclastogenesis is comprised of several stage s including progenitor survival, differentiation to mononuclear preosteoclasts, cell fusion to multinuclear mature osteoclasts, and activation to osteoclasts with bone resorbing activity. Botanical antioxidants are now being increasingly investigated for their health-promoting effects on bone. This study investigated that fisetin, a flavonol found naturally in many fruits and vegetables, suppressed osteoclastogenesis by disturbing receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated signaling pathway and demoting osteoclastogenic protein induction. Nontoxic fisetin at ≤10 µM inhibited the induction of RANK, tumor necrosis factor receptor associated factor 6 (TRAF6) and the activation of NF-κB in RANKL-stimulated RAW 264.7 macrophages. In RANKL-differentiated osteoclasts cell fusion protein of E-cadherin was induced, which was dampened by fisetin. The formation of tartrate-resistance acid phosphatase-positive multinucleated osteoclasts was suppressed by adding fisetin to RANKL-exposed macrophages. It was also found that fisetin reduced actin ring formation and gelsolin induction of osteclasts enhanced by RANKL through disturbing c-Src-proline-rich tyrosine kinase 2 signaling. Fisetin deterred preosteoclasts from the cell-cell fusion and the organization of the cytoskeleton to seal the resorbing area and to secret protons for bone resorption. Consistently, the 5 day-treatment of fisetin diminished RANKL-induced cellular expression of carbonic anhydrase II and integrin ß3 concurrently with a reduction of osteoclast bone-resorbing activity. Therefore, fisetin was a natural therapeutic agent retarding osteoclast fusion and cytoskeletal organization such as actin rings and ruffled boarder, which is a property of mature osteoclasts and is required for osteoclasts to resorb bone.

A medium-chain fatty acid, capric acid, inhibits RANKL-induced osteoclast differentiation via the suppression of NF-κB signaling and blocks cytoskeletal organization and survival in mature osteoclasts.

Fatty acids, important components of a normal diet, have been reported to play a role in bone metabolism. Osteoclasts are bone-resorbing cells that are responsible for many bone-destructive diseases such as osteoporosis. In this study, we investigated the impact of a medium-chain fatty acid, capric acid, on the osteoclast differentiation, function, and survival induced by receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (MCSF). Capric acid inhibited RANKL-mediated osteoclastogenesis in bone marrow-derived macrophages and suppressed RANKL-induced IκBα phosphorylation, p65 nuclear translocation, and NF-κB transcriptional activity. Capric acid further blocked the RANKL-stimulated activation of ERK without affecting JNK or p38. The induction of NFATc1 in response to RANKL was also attenuated by capric acid. In addition, capric acid abrogated M-CSF and RANKL-mediated cytoskeleton reorganization, which is crucial for the efficient bone resorption of osteoclasts. Capric acid also increased apoptosis in mature osteoclasts through the induction of Bim expression and the suppression of ERK activation by M-CSF. Together, our results reveal that capric acid has inhibitory effects on osteoclast development. We therefore suggest that capric acid may have potential therapeutic implications for the treatment of bone resorption-associated disorders.