Three-dimensional Collagen Gel Research Articles

Depletion of Ras Suppressor-1 (RSU-1) promotes cell invasion of breast cancer cells through a compensatory upregulation of a truncated isoform

Extracellular matrix (ECM)-adhesion proteins and actin cytoskeleton are pivotal in cancer cell invasion. Ras Suppressor-1 (RSU-1), a cell-ECM adhesion protein that interacts with PINCH-1, thus being connected to Integrin Linked Kinase (ILK), alpha-parvin (PARVA), and actin cytoskeleton, is up-regulated in metastatic breast cancer (BC) samples. Apart from the originally-identified gene (RSU-1L), an alternatively-spliced isoform (RSU-1-X1) has been reported. We used non-invasive MCF-7 cells, expressing only RSU-1L, and highly invasive MDA-MB-231-LM2 expressing both isoforms and generated stable shRNA-transduced cells lacking RSU-1L, while the truncated RSU-1-X1 isoform was depleted by siRNA-mediated silencing. RSU-1L depletion in MCF-7 cells resulted in complete abrogation of tumor spheroid invasion in three-dimensional collagen gels, whereas it promoted MDA-MB-231-LM2 invasion, through a compensatory upregulation of RSU-1-X1. When RSU-1-X1 was also eliminated, RSU-1L-depletion-induced migration and invasion were drastically reduced being accompanied by reduced urokinase plasminogen activator expression. Protein expression analysis in 23 human BC samples corroborated our findings showing RSU-1L to be upregulated and RSU-1-X1 downregulated in metastatic samples. We demonstrate for the first time, that both RSU-1 isoforms promote invasion in vitro while RSU-1L elimination induces RSU-1-X1 upregulation to compensate for the loss. Hence, we propose that both isoforms should be blocked to effectively eliminate metastasis.

Depletion of glycosphingolipids induces excessive response of chondrocytes under mechanical stress

Glycosphingolipids (GSLs) are ubiquitous membrane components that play an indispensable role in maintaining chondrocyte homeostasis. To gain better insight into roles of GSLs, we studied the effects of GSL-deletion on the physiological responses of chondrocytes to mechanical stress. Mice lacking Ugcg gene (Ugcg-/-) were genetically generated to obtain GSL-deficient mice, and their chondrocytes from the joints were used for functional analyses in vitro culture experiments. The cells were seeded in a three-dimensional collagen gel and subjected to 5%, 10% or 16% cyclic tensile strain for either 3 or 24 h. The gene expressions of chondrocyte anabolic and catabolic factors, and the induction of Ca2+ signaling were analyzed. Our results revealed that chondrocytes derived from GSL-deficient mice exhibited an elevation in the expression of catabolic factors (ADAMTS-5, MMP-13) following the exposure to strain with amplitudes of 10%. Likewise, applying cyclic tensile strain with these amplitudes resulted in an increased Ca2+ oscillation ratio in chondrocytes from GSL-deficient as compared to the ratio from control mice. These results demonstrated that deletion of GSL stimulated the catabolic responses of chondrocytes to mechanical stress via the augmentation of the sensitivity to mechanical stress that may lead to the cartilage deterioration. These findings suggest that the regulation of the physiological responses of chondrocytes by GSLs could be a potential target in a therapeutic intervention in osteoarthritis.

Inhibition by Epigallocatechin Gallate of IL-1-Induced Urokinase-Type Plasminogen Activator Expression and Collagen Degradation by Corneal Fibroblasts.

The proinflammatory cytokine interleukin (IL)-1 is implicated in corneal ulceration and promotes collagen degradation by corneal fibroblasts cultured in a three-dimensional (3D) collagen gel. Epigallocatechin-3-gallate (EGCG), the principal polyphenol in extracts of green tea, has various beneficial health effects, some of which appear to be mediated through direct or indirect inhibition of protease activity. We therefore examined the effect of EGCG on IL-1β-induced collagen degradation by corneal fibroblasts embedded in a collagen gel. Human corneal fibroblasts were cultured in a type I collagen gel. Collagen degradation was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. The expression of urokinase-type plasminogen activator (uPA) was examined by real-time and RT-PCR analysis and by fibrin zymography, and that of the collagenase matrix metalloproteinase 1 (MMP1) was detected by immunoblot analysis. EGCG inhibited IL-1β-induced, plasminogen-dependent collagen degradation by corneal fibroblasts in a concentration-dependent manner. It also attenuated the IL-1β-induced expression of uPA at both mRNA and protein levels. EGCG inhibited the IL-1β-induced conversion of exogenous plasminogen to plasmin as well as the plasminogen-dependent activation of pro-MMP1 in the 3D cultures without a substantial effect on pro-MMP1 abundance. EGCG inhibits IL-1β-induced collagen degradation by corneal fibroblasts, with this effect likely being mediated by suppression of the upregulation of uPA, the uPA-mediated conversion of plasminogen to plasmin, and the plasmin-mediated activation of pro-MMP1. EGCG thus warrants further investigation as a potential treatment for corneal ulcer.

Tenomodulin regulates matrix remodeling of mouse tendon stem/progenitor cells in an ex vivo collagen I gel model

Tenomodulin (Tnmd) is predominantly expressed in tendon and ligament tissues. Loss of Tnmd in mice leads to a profound phenotype in vitro, characterized by reduced self-renewal but increased senescence of mouse tendon stem/progenitor cells (mTSPCs), as well as in vivo, by significantly impaired early tendon healing. Interestingly, injuried Achilles tendons from Tnmd-deficient mice showed inferior tendon repair, which was characterized by less contracted fibrovascular scars with disorganized matrix composition in comparison to wild type (WT) mice at day 8 after injury. To better understand Tnmd role in tendon repair, here we implemented an ex vivo three-dimensional (3D) collagen gel model and investigated whether Tnmd knockout affects the collagen contraction of mTSPCs. TSPCs were isolated from WT and Tnmd knockout (KO) tendons at 6, 9, 12, and 18 months of age. Adhesion assay demonstrated that loss of Tnmd in mTSPCs resulted in reduced adhesion to collagen type I. Quantitative time-dependent analysis revealed that Tnmd-deficient mTSPCs of all ages have significantly reduced capacity to contract collagen matrix in comparison to WT cells. Furthermore, 18 months old mTSPCs of both genotypes showed lower collagen contractility than cells obtained from 6, 9, and 12 months old animals, demonstrating an overall effect of organismal aging on matrix remodeling. Nevertheless, both cell types had a similar survival rate for the 5 days of cultivation within the gels. Lastly, quantitative PCR for 48 different genes revealed that the knockout of Tnmd majorly affected the gene expression profile of mTSPCs, as several transcription factors, tendon matrix, collagen cross-linking, and lineage maker genes were down-regulated. Taken together, our results clearly demonstrated that loss of Tnmd in mTSPCs led to profoundly altered gene expression profile, insufficient adhesion to collagen type I, and impaired ability to contract the extracellular matrix.

Breast Cancer Cells Adapt Contractile Forces to Overcome Steric Hindrance

Cell migration through the extracellular matrix is governed by the interplay between cell-generated propulsion forces, adhesion forces, and resisting forces arising from the steric hindrance of the matrix. Steric hindrance in turn depends on matrix porosity, matrix deformability, cell size, and cell deformability. In this study, we investigate how cells respond to changes in steric hindrance that arise from altered cell mechanical properties. Specifically, we measure traction forces, cell morphology, and invasiveness of MDA-MB 231 breast cancer cells in three-dimensional collagen gels. To modulate cell mechanical properties, we either decrease nuclear deformability by twofold overexpression of the nuclear protein lamin A or we introduce into the cells stiff polystyrene beads with a diameter larger than the average matrix pore size. Despite this increase of steric hindrance, we find that cell invasion is only marginally inhibited, as measured by the fraction of motile cells and the mean invasion depth. To compensate for increased steric hindrance, cells employ two alternative strategies. Cells with higher nuclear stiffness increase their force polarity, whereas cells with large beads increase their net contractility. Under both conditions, the collagen matrix surrounding the cells stiffens dramatically and carries increased strain energy, suggesting that increased force polarity and increased net contractility are functionally equivalent strategies for overcoming an increased steric hindrance.

Dynamic Histological Events and Molecular Changes in Excisional Wound Healing of Diabetic DB/DB Mice

BackgroundWound contraction and re-epithelialization over the entire healing process had never been histologically examined daily in diabetic mouse wounds. Correlating morphological characters with molecular changes may be essential to understand the potential mechanism of impeded diabetic wound healing. Materials and methodsIn 99 db/db and 63 db/m mice, dorsal-paired 8 mm-diameter wounds were created. Wound contraction and re-epithelialization were histologically analyzed daily—six wounds per group each day. A novel three-dimensional collagen gel model was used to study diabetic dermal fibroblast contractility. Fibroblast-to-myofibroblasts differentiation and TGFβ-SMAD signaling pathway through the diabetic db/db wound healing process were studied by immunohistochemistry. ResultsDb/db wounds presented delayed closure with impaired wound contraction. Re-epithelialization was not slow but showed thinner epithelial formation and irregular keratinocyte arrangement. Diabetic dermal fibroblasts had significantly lower contractile ability than nondiabetic fibroblasts. In db/db wounds, α-SMA, the marker of myofibroblasts, showed constantly low through the healing, which represented reduced fibroblast-to-myofibroblasts differentiation. Remarkably weak staining of TGFβRI and low accumulation of Smad3 in nuclei were observed. ConclusionsWe demonstrated and precisely located downregulated TGFβ signaling pathway in db/db wounds by showing low expression of TGFβRI and failure of Smad3 translocation from cytoplasm to nuclei, which was not reported previously. The downregulated TGFβ signaling pathway may contribute to the attenuated fibroblast-to-myofibroblast differentiation. Deficient re-epithelialization and defective wound contraction contribute principally to delayed healing of diabetic db/db wounds.

Simple and cost-effective assay for isolating invasive living cells.

A modified invasion assay using a three-dimensional collagen gel was developed that enables isolation of invasive living cells; it was named the invading cell trapping (iCT) assay. A small cell strainer consisting of a nylon mesh with 40-μm2 pores was used, and collagen gel layers formed across the membrane. Test cells were seeded in the lower gel layer and invasive cells were attracted upward and trapped in the upper gel. After incubation, the collagen gel layers in cell strainers were easily separated and living cells in the gel were counted and analyzed. An advantage of the iCT assay is that it can capture living invasive cells in the upper gel while leaving noninvasive ones in the lower layer. Further enrichment of the two cell populations can be achieved by repeating the assay. Thus, the iCT assay allows comparative analysis of invasive versus noninvasive cells.

Assessing fluorescence detection and effective photothermal therapy of near-infrared polymer nanoparticles using alginate tissue phantoms.

Photothermal therapy (PTT) uses light absorbing materials to generate heat for treatment of diseases, like cancer. The advantages of using PTT components that absorb in the near-infrared (NIR) include reduced tissue auto-fluorescence and higher penetration depths. However, NIR laser light can still be scattered and absorbed by biological tissues, thus decreasing the amount of the energy reaching the PTT agents. We have developed two distinct formulations of NIR-absorbing nanoparticles, one which can be utilized for PTT only, and another for both PTT and fluorescence imaging of colorectal cancer. In this work, the fluorescence detection limit and the PTT heating potential of the two nanoparticle types were determined using alginate tissue phantoms. The objective of this study was to determine the PTT efficiency and theranostic potential of the nanoparticles by irradiating 3D collagen tumor spheroids, containing nanoparticles and CT26 mouse colorectal cancer cells, through increasing tissue phantom thicknesses and then quantifying cell death. Materials and Methods Our lab has previously developed nanoparticles based on the semiconducting, conjugated polymer poly[4,4-bis(2-ethylhexyl)-cyclopenta[2,1-b;3,4-b']dithiophene-2,6-diyl-alt-2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe). We have also made a hybrid nanoparticle that heats and fluoresces by combining PCPDTBSe polymer with the fluorescent poly[(9,9-dihexylfluorene)-co-2,1,3-benzothiadiazole-co-4,7-di(thiophen-2-yl)-2,1,3-benzothiadiazole] (PFBTDBT10) polymer to yield nanoparticles termed Hybrid Donor-Acceptor Polymer Particles (H-DAPPs). H-DAPPs and PCPDTBSe nanoparticles were added to three-dimensional collagen gel tumor spheroids in order to represent nanoparticles in a tumor. Alginate tissue phantoms, comprised of an optical scattering agent (Intralipid) and an optical absorbing material (hemoglobin) in order to mirror biological tissue scattering effects, were used to simulate increasing tissue thickness between the nanoparticles and the PTT energy source. Fluorescence from the H-DAPPs was detectable through 6 mm of tissue phantoms. It was found that less than 10% of the laser energy could penetrate through 9 mm of tissue phantoms and only 60% of the laser energy passed through the 1.5 mm phantoms, regardless of laser power. PTT experiments, using 800 nm light at 2.2 W/cm2 for 60 s through tissue phantoms to stimulate nanoparticle-doped tumor spheroids, showed 55% cell death through 3 mm of tissue phantoms using H-DAPPs. Results from using the PCPDTBSe nanoparticles showed 72% cell death through 3 mm and over 50% cell death through 6 mm of tissue phantoms. The results of this work validated the heating potential and fluorescence detection limitations of two theranostic polymer nanoparticles by utilizing alginate tissue phantoms and 3D tumor spheroids. H-DAPPs and PCPDTBSe polymer nanoparticles can be utilized as effective PTT agents by exploiting their absorption of NIR light and H-DAPPs have advantageous fluorescence for imaging colorectal cancer. The data generated from this study design can allow for other NIR absorbing and fluorescing nanoparticle formulations to be evaluated prior to in vivo experimentation. Lasers Surg. Med. 50:1040-1049, 2018. © 2018 Wiley Periodicals, Inc.

Similar Biophysical Abnormalities in Glomeruli and Podocytes from Two Distinct Models.

Background FSGS is a pattern of podocyte injury that leads to loss of glomerular function. Podocytes support other podocytes and glomerular capillary structure, oppose hemodynamic forces, form the slit diaphragm, and have mechanical properties that permit these functions. However, the biophysical characteristics of glomeruli and podocytes in disease remain unclear.Methods Using microindentation, atomic force microscopy, immunofluorescence microscopy, quantitative RT-PCR, and a three-dimensional collagen gel contraction assay, we studied the biophysical and structural properties of glomeruli and podocytes in chronic (Tg26 mice [HIV protein expression]) and acute (protamine administration [cytoskeletal rearrangement]) models of podocyte injury.Results Compared with wild-type glomeruli, Tg26 glomeruli became progressively more deformable with disease progression, despite increased collagen content. Tg26 podocytes had disordered cytoskeletons, markedly abnormal focal adhesions, and weaker adhesion; they failed to respond to mechanical signals and exerted minimal traction force in three-dimensional collagen gels. Protamine treatment had similar but milder effects on glomeruli and podocytes.Conclusions Reduced structural integrity of Tg26 podocytes causes increased deformability of glomerular capillaries and limits the ability of capillaries to counter hemodynamic force, possibly leading to further podocyte injury. Loss of normal podocyte mechanical integrity could injure neighboring podocytes due to the absence of normal biophysical signals required for podocyte maintenance. The severe defects in podocyte mechanical behavior in the Tg26 model may explain why Tg26 glomeruli soften progressively, despite increased collagen deposition, and may be the basis for the rapid course of glomerular diseases associated with severe podocyte injury. In milder injury (protamine), similar processes occur but over a longer time.

Atomic force microscopy nano-characterization of 3D collagen gels with tunable stiffness

As extracellular matrix (ECM) nano-characteristics play a crucial role in cell behavior, including cancer development and metastasis, several ECM in vitro models have been used in order to study cells behavior under different biochemical and mechanical conditions. Among the ECM constituents, collagen (especially collagen type I) has been extensively used as an essential component of ECM models, since it is one of the most abundant ECM protein. Use of three-dimensional (3D) collagen gels provides the advantage of allowing the cells to grow in a 3D environment that bears strong similarities to their natural, in vivo setting. Thus, the ability to form collagen gels with tunable stiffness and well defined naturally occurring nano-characteristics is crucial for these studies. Atomic Force Microscopy (AFM) is a unique tool that is ideal for the complete characterization of such models, in terms of morphology and mechanical properties without destroying the collagen fiber structure. In this protocol, the development and the AFM nano-scale characterization of 3D collagen type I gels is presented. The protocol includes:•The formation of 3D collagen type I gels with tunable stiffness•The preparation of histological sections from collagen gels•The AFM-based morphological and mechanical nano-characterization of the gels

ATM Is Required for the Prolactin-Induced HSP90-Mediated Increase in Cellular Viability and Clonogenic Growth After DNA Damage.

Prolactin (PRL) acts as a survival factor for breast cancer cells, but the PRL signaling pathway and the mechanism are unknown. Previously, we identified the master chaperone, heat shock protein 90 (HSP90) α, as a prolactin-Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) target gene involved in survival, and here we investigated the role of HSP90 in the mechanism of PRL-induced viability in response to DNA damage. The ataxia-telangiectasia mutated kinase (ATM) protein plays a critical role in the cellular response to double-strand DNA damage. We observed that PRL increased viability of breast cancer cells treated with doxorubicin or etoposide. The increase in cellular resistance is specific to the PRL receptor, because the PRL receptor antagonist, Δ1-9-G129R-hPRL, prevented the increase in viability. Two different HSP90 inhibitors, 17-allylamino-17-demethoxygeldanamycin and BIIB021, reduced the PRL-mediated increase in cell viability of doxorubicin-treated cells and led to a decrease in JAK2, ATM, and phosphorylated ATM protein levels. Inhibitors of JAK2 (G6) and ATM (KU55933) abolished the PRL-mediated increase in cell viability of DNA-damaged cells, supporting the involvement of each, as well as the crosstalk of ATM with the PRL pathway in the context of DNA damage. Drug synergism was detected between the ATM inhibitor (KU55933) and doxorubicin and between the HSP90 inhibitor (BIIB021) and doxorubicin. Short interfering RNA directed against ATM prevented the PRL-mediated increase in cell survival in two-dimensional cell culture, three-dimensional collagen gel cultures, and clonogenic cell survival, after doxorubicin treatment. Our results indicate that ATM contributes to the PRL-JAK2-STAT5-HSP90 pathway in mediating cellular resistance to DNA-damaging agents.

IGF-1 promotes angiogenesis in endothelial cells/adipose-derived stem cells co-culture system with activation of PI3K/Akt signal pathway.

The aim of this study was to investigate the role of insulin-like growth factor-1 (IGF-1) and crosstalk between endothelial cells (ECs) and adipose-derived stem cells (ASCs) in the process of angiogenesis. A three-dimensional collagen gel used to culture mouse ASCs and mouse ECs in vitro was established. The effects of angiogenesis after exposure to IGF-1 were observed by confocal laser scanning microscopy. Western blotting and qPCR were performed to elucidate the underlying mechanisms. IGF-1 treatment promoted the formation of vessel-like structures and the recruitment of ASCs in the three-dimensional collagen gel. The angiogenic genes and proteins in ECs were up-regulated by IGF-1 and in co-culture. Similar changes in the genes and in the proteins were detected in ASCs after exposure to IGF-1 and co-culture. p-Akt expression levels were high in ECs and ASCs after exposure to IGF-1 and co-culture. IGF-1 and co-culture between cells facilitate the process of angiogenesis via the PI3-kinase/Akt signalling pathway. In ECs, IGF-1 stimulates the expression of angiogenesis-related growth factors with the activation of the PI3-kinase/Akt signalling pathway. Co-cultured ECs exposed to excess VEGF-A and other angiogenesis-related growth factors para-secreted from ASCs exhibit high expression of angiogenesis-related genes and proteins. In ASCs, IGF-1 induces the recruitment and function of ASCs by up-regulating the expression of PDGFB, MMPs and α-SMA. Crosstalk with ECs further facilitates changes in ASCs.

Role of macrophage migration inhibitory factor (MIF) in the effects of oxidative stress on human retinal pigment epithelial cells

Proliferative vitreoretinopathy (PVR) is the major cause of treatment failure in individuals who undergo surgery for retinal detachment. The epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells contributes to the pathogenesis of PVR. Oxidative stress is thought to play a role in the progression of retinal diseases including PVR. We have now examined the effects of oxidative stress on the EMT and related processes in the human RPE cell line. We found that H2 O2 induced the contraction of RPE cells in a three-dimensional collagen gel. Analysis of a cytokine array revealed that H2 O2 specifically increased the release of macrophage migration inhibitory factor (MIF) from RPE cells. Reverse transcription-polymerase chain reaction and immunoblot analyses showed that H2 O2 increased the expression of MIF in RPE cells. Immunoblot and immunofluorescence analyses revealed that H2 O2 upregulated the expression of α-SMA and vimentin and downregulated that of ZO-1 and N-cadherin. Consistent with these observations, the transepithelial electrical resistance of cell was reduced by exposure to H2 O2 . The effects of oxidative stress on EMT-related and junctional protein expression as well as on transepithelial electrical resistance were inhibited by antibodies to MIF, but they were not mimicked by treatment with recombinant MIF. Finally, analysis with a profiling array for mitogen-activated protein kinase signalling revealed that H2 O2 specifically induced the phosphorylation of p38 mitogen-activated protein kinase. Our results thus suggest that MIF may play a role in induction of the EMT and related processes by oxidative stress in RPE cells and that it might thereby contribute to the pathogenesis of PVR. Proliferative vitreoretinopathy is a major complication of rhegmatogenous retinal detachment, and both oxidative stress and induction of the EMT in RPE cells are thought to contribute to the pathogenesis of this condition. We have now examined the effects of oxidative stress on the EMT and related processes in the human RPE cell line ARPE19. Our results thus implicate MIF in induction of the EMT and related processes by oxidative stress in RPE cells and the regulated expression of EMT markers. They further suggest that MIF may play an important role in the pathogenesis of PVR.

Post-translational modification of the membrane type 1 matrix metalloproteinase (MT1-MMP) cytoplasmic tail impacts ovarian cancer multicellular aggregate dynamics

Membrane type 1 matrix metalloproteinase (MT1-MMP, MMP-14) is a transmembrane collagenase highly expressed in metastatic ovarian cancer and correlates with poor survival. Accumulating evidence shows that the cytoplasmic tail of MT1-MMP is subjected to phosphorylation, and this post-translational modification regulates enzymatic activity at the cell surface. To investigate the potential role of MT1-MMP cytoplasmic residue Thr567 phosphorylation in regulation of metastasis-associated behaviors, ovarian cancer cells that express low endogenous levels of MT1-MMP were engineered to express wild-type MT1-MMP, a phosphomimetic mutant (T567E), or a phosphodeficient mutant (T567A). Results show that Thr567 modulation influences behavior of both individual cells and multicellular aggregates (MCAs). The acquisition of either wild-type or mutant MT1-MMP expression results in altered cohesion of epithelial sheets and the formation of more compact MCAs relative to parental cells. Cells expressing MT1-MMP-T567E phosphomimetic mutants exhibit enhanced cell migration. Furthermore, MCAs formed from MT1-MMP-T567E-expressing cells adhere avidly to both intact ex vivo peritoneal explants and three-dimensional collagen gels. Interaction of these MCAs with peritoneal mesothelium disrupts mesothelial integrity, exposing the submesothelial collagen matrix on which MT1-MMP-T567E MCAs rapidly disperse. Together, these findings suggest that post-translational regulation of the Thr567 in the MT1-MMP cytoplasmic tail may function as a regulatory mechanism to impact ovarian cancer metastatic success.

Activation of Parathyroid Hormone 2 Receptor Induces Decorin Expression and Promotes Wound Repair

In this study, we report that TIP39, a parathyroid hormone ligand family member that was recently identified to be expressed in the skin, can induce decorin expression and enhance wound repair. Topical treatment of mice with TIP39 accelerated wound repair, whereas TIP39-deficient mice had delayed repair that was associated with formation of abnormal collagen bundles. To study the potential mechanism responsible for the action of TIP39 in the dermis, fibroblasts were cultured in three-dimensional collagen gels, a process that results in enhanced decorin expression unless activated to differentiate to adipocytes, whereupon these cells reduce expression of several proteoglycans, including decorin. Small interfering RNA-mediated silencing of parathyroid hormone 2 receptor (PTH2R), the receptor for TIP39, suppressed the expression of extracellular matrix-related genes, including decorin, collagens, fibronectin, and matrix metalloproteases. Skin wounds in TIP39-/- mice had decreased decorin expression, and addition of TIP39 to cultured fibroblasts induced decorin and increased phosphorylation and nuclear translocation of CREB. Fibroblasts differentiated to adipocytes and treated with TIP39 also showed increased decorin and production of chondroitin sulfate. Furthermore, the skin of PTH2R-/- mice showed abnormal extracellular matrix structure, decreased decorin expression, and skin hardness. Thus, the TIP39-PTH2R system appears to be a previously unrecognized mechanism for regulation of extracellular matrix formation and wound repair.

Suppression by an RAR-γ Agonist of Collagen Degradation Mediated by Corneal Fibroblasts

To examine the role of retinoic acid receptor (RAR) isoforms in interleukin-1β (IL-1β)-induced collagen degradation by corneal fibroblasts. Primary rabbit corneal fibroblasts embedded in a three-dimensional collagen gel were incubated with or without all-trans retinoic acid (ATRA), the RAR-α agonist Am580, the RAR-β agonist AC55649, or the RAR-γ agonist R667. Collagen degradation was determined by measurement of hydroxyproline produced in acid hydrolysates of culture supernatants. Matrix metalloproteinase (MMP) expression was evaluated by immunoblot analysis and gelatin zymography. The phosphorylation of mitogen-activated protein kinases (MAPKs) and the endogenous nuclear factor (NF)-κB inhibitor IκB-α was examined by immunoblot analysis. Cell proliferation was measured with a bromodeoxyuridine incorporation assay, and cell viability was determined by measurement of the release of lactate dehydrogenase. Interleukin-1β-induced collagen degradation by corneal fibroblasts was inhibited by ATRA, Am580, and R667 in a concentration-dependent manner but was unaffected by AC55649, with the inhibitory effects of ATRA and R667 being markedly greater than that of Am580. The IL-1β-induced production of MMP-1, MMP-2, MMP-3, and MMP-9 by corneal fibroblasts was also inhibited by R667 in a concentration-dependent manner. R667 inhibited the IL-1β-induced phosphorylation of IκB-α but not that of MAPKs. R667 had no effect on the proliferation or viability of corneal fibroblasts. The RAR-γ agonist R667 suppressed MMP production and thereby inhibited collagen degradation by corneal fibroblasts exposed to the proinflammatory cytokine IL-1β. These effects of R667 may be mediated by the NF-κB signaling pathway.

Focal adhesion kinase signaling determines the fate of lung epithelial cells in response to TGF-β

Alveolar epithelial cell (AEC) injury and apoptosis are prominent pathological features of idiopathic pulmonary fibrosis (IPF). There is evidence of AEC plasticity in lung injury repair response and in IPF. In this report, we explore the role of focal adhesion kinase (FAK) signaling in determining the fate of lung epithelial cells in response to transforming growth factor-β1 (TGF-β1). Rat type II alveolar epithelial cells (RLE-6TN) were treated with or without TGF-β1, and the expressions of mesenchymal markers, phenotype, and function were analyzed. Pharmacological protein kinase inhibitors were utilized to screen for SMAD-dependent and -independent pathways. SMAD and FAK signaling was analyzed using siRNA knockdown, inhibitors, and expression of a mutant construct of FAK. Apoptosis was measured using cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. TGF-β1 induced the acquisition of mesenchymal markers, including α-smooth muscle actin, in RLE-6TN cells and enhanced the contraction of three-dimensional collagen gels. This phenotypical transition or plasticity, epithelial-myofibroblast plasticity (EMP), is dependent on SMAD3 and FAK signaling. FAK activation was found to be dependent on ALK5/SMAD3 signaling. We observed that TGF-β1 induces both EMP and apoptosis in the same cell culture system but not in the same cell. While blockade of SMAD signaling inhibited EMP, it had a minimal effect on apoptosis; in contrast, inhibition of FAK signaling markedly shifted to an apoptotic fate. The data support that FAK activation determines whether AECs undergo EMP vs. apoptosis in response to TGF-β1 stimulation. TGF-β1-induced EMP is FAK- dependent, whereas TGF-β1-induced apoptosis is favored when FAK signaling is inhibited.

The selective cysteinyl leukotriene receptor 1 (CysLT1R) antagonist montelukast regulates extracellular matrix remodeling

Scar formation after filtration surgery of glaucoma is mainly caused by excessive synthesis of new extracellular matrix (ECM) and contraction of subconjunctival tissue mediated by human Tenon fibroblasts (HTFs) and the transforming growth factor (TGF-β1). Montelukast, a potent and specific cysteinyl leukotriene receptor 1 (cysLT1R) antagonist, is a licensed drug clinically used for the treatment of bronchial asthma. In this study, we investigated the effects of montelukast on the contractility of HTFs cultured in a three-dimensional collagen gel. We found that cysLT1R was expressed in HTFs. Interestingly, the expression of cysLT1R was increased in response to TGF-β1 in a dose dependent manner, suggesting its potential role in TGF-β1 induced fibrosis. Importantly, we found that montelukast inhibited TGF-β1-induced collagen gel contraction mediated by HTFs in a concentration- and time-dependent manner. In addition, TGF-β1-induced expression of MMP-1 and MMP-3, generation of fibronectin and type I collagen production, focal adhesion kinase (FAK) and paxillin phosphorylation in HTFs were also ameliorated by montelukast in a dose dependent manner. These results suggested that montelukast might provide therapeutic possibilities for inhibition of scar formation after such surgery.

Notch Signaling Pathway Regulates Angiogenesis via Endothelial Cell in 3D Co-Culture Model.

This study aimed to investigate the role of Notch signaling pathway for angiogenesis in a three-dimensional (3D) collagen gel model with co-culture of adipose-derived stromal cells (ASCs) and endothelial cells (ECs). A 3D collagen gel model was established in vitro by implanting both ASCs from green fluorescent protein-labeled mouse and ECs from red fluorescent protein-labeled mouse, and the phenomena of angiogenesis with Notch signaling inducer Jagged1, inhibitor DAPT and PBS, respectively were observed by confocal laser scanning microscopy. Semi-quantitative PCR and immunofluorescent staining were conducted to detect expressions of angiogenesis-related genes and proteins. Angiogenesis in the co-culture gels was promoted by Jagged1 treatment while attenuated by DAPT treatment, compared to control group. In co-culture system of ASCs and ECs, the gene expressions of VEGFA, VEGFB, Notch1, Notch2, Hes1, Hey1, VEGFR1,and the protein expression of VEGFA, VEGFB, Notch1, Hes1, Hey1 were increased by Jagged1 treatment and decreased by DAPT treatment in ECs. And the result of VEGFR3 was the opposite. However, the same results did not appear completely in ASCs. These results revealed the VEGFA/B-Notch1/2-Hes1/Hey1- VEGFR1/3 signal axis played an important role in angiogenesis when ASCs and ECs were co-cultured in a 3D collagen gel model. J. Cell. Physiol. 232: 1548-1558, 2017. © 2016 Wiley Periodicals, Inc.

Cooperative cell invasion: matrix metalloproteinase-mediated incorporation between cells.

Progression to metastatic disease is a leading cause of cancer death. Tumors are a complex mixture of cell types, both genetically heterogeneous malignant cells and associated nonmalignant cells. Models mimicking this heterogeneous cell environment have revealed that invasive cell populations can induce dissemination by otherwise poorly/noninvasive tumor cells, known as cooperative invasion. Neuroblastoma tumors arise in children and are characterized by mixed cellular populations in vivo, consisting chiefly of neuronal (N)-type and substrate (S)-type cells. The S-type cells have all the hallmarks of invasive leader cell populations and have been coisolated with N-type cells from metastatic bone lesions, but to date their ability to induce cooperative invasion has not been investigated. Therefore, in the present study, we analyzed the invasive behavior of mixed N-type and S-type multicellular spheroids embedded in three-dimensional collagen gels. Our analyses show that S-type cells induce invasion of either single cells or small cell clusters of N-type cells. In contrast to other reports of cooperative invasion in which mixed cultures exhibit a follow-the-leader mechanism, we show coincident emergence of S- and N-type cells from mixed spheroids. Our data suggest mutual effects between the two cell types. Thus, whereas coculture with S-type cells induces N-type invasion, coculture with N-type cells slows S-type invasion. Using matrix metalloproteinase (MMP) inhibitors and cell incorporation assays, we demonstrate that MMP activity is required for S-type cells to insert into layers of N-type cells. Our study therefore highlights an important role for S-type neuroblastoma cells in the invasion process and reveals a new mechanism of cooperative invasion.