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559 Beneficial effects of Silybum marianum extract and silymarin on regulation of decorin and biglycan
ABSTRACTS | Growth Factors, Cell Adhesion and Matrix Biology 554
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Dynamic organization and order of desmoglein 3 in desmosomes determined by fluorescence polarization microscopy EI Bartle, TM Urner and AL Mattheyses Emory University School of Medicine, Atlanta, GA Desmosomes are macromolecular cell-cell complexes responsible for providing strong adhesion between epidermal keratinocytes by coupling extracellular adhesions to the intracellular intermediate filament cytoskeleton. Electron microscopy studies have revealed that the cadherin proteins in the adhesive core of desmosomes appear to be organized.However,due to the size and molecular complexity of desmosomes, how cadherin order relates to adhesion in living cells has been challenging to study. We addressed this challenge by developing and applying fluorescence polarization microscopy to study protein order in desmosomes. Fluorescence polarization microscopy allows characterization of collective protein order or disorder within macromolecular complexes. The cadherin desmoglein 3 was tagged with GFP (Dsg3-DEA-GFP) and transfected into HaCaT human keratinocyte cells. Imaging with fluorescence polarization microscopy revealed that the extracellular domain of desmoglein 3 is ordered in desmosomes in living cells. To investigate if this order is correlated to adhesive function, calcium was depleted from the media of HaCaT cells with calcium sensitive desmosomes. This led to a progressive loss of adhesive strength over 30 minutes, as determined by a cell fragmentation assay. Time courses of individual cells imaged with fluorescence polarization revealed a concurrent loss of Dsg3-DEA-GFP order. Our results establish that the order and organization of the desmosomal cadherins can be measured in living cells. We propose that extracellular domain order can be used as a optical biomarkerallowing spatio-temporal measurement of adhesive strength in individual desmosomes. These results demonstrate that fluorescence polarization microscopy is a novel approach to study desmosome organization and function in living cells and in human disease.
Amphiregulin silencing overcomes resistance to the EGF receptor inhibitor cetuximab in head and neck squamous cell carcinoma SW Stoll1 and J Elder2 1 University of Michigan, Ann Arbor, MI and 2 Dept. of Dermatology, U.of Michigan, Ann Arbor, MI EGF receptor (EGFR) is central to epithelial cell physiology and its deregulated signaling has been implicated in the pathogenesis of various epithelial neoplasias. Overexpression of EGFR is an important prognostic factor in head and neck squamous cell carcinoma (HNSCC) and EGFR blockade has emerged as a significant yet imperfect treatment modality for these tumors. In human skin keratinocytes (KC) we have previously shown that interfering with EGFR signaling through silencing of the EGFR ligand amphiregulin (AREG) leads to a pronounced state of G2/M/cytokinesis growth arrest that cannot be reversed by addition of exogenous EGFR ligands, including AREG itself. To compare the efficacy of AREG silencing and EGFR blockade in human skin KC and malignant epithelial cells we treated the HNSCC cell lines UMSCC-22A and UMSCC-22B, which are derived from a primary HNSCC and its metastasis, with the EGFR inhibitor cetuximab. Incubation of these cells with various concentrations of cetuximab led to a reduction in cell numbers which was much more modest than in immortalized but non-transformed human N/TERT-KC (UMSCC-22A: 45.2% of control at 500 ng/ml and 42.5% at 1000 ng/ml; UMSCC-22B: 46.9% and 35.4%; N/TERT: 18.1% and 11.1%; n¼2-4). In contrast, and very similar to N/TERT-KC (JID 136:444), shRNA-mediated AREG silencing reduced HNSCC cell growth by more than 88% (UMSCC-22A: 12 +/- 2.3% of control; UMSCC-22B 11.5 +/- 1.1%, mean +/- SEM, n¼4). Because of the already strong inhibition of cell growth in response to AREG silencing, a combination of cetuximab and AREG shRNA resulted only in a slight further improvement. Similar to N/TERT-KC, AREG silencing in these HNSCC cell lines led to the appearance of numerous bi- and multinucleated cells, many of which appeared to be undergoing mitotic catastrophe. Together, our findings suggest that UMSCC-22A and UMSCC-22B cells are similarly sensitive to AREG silencing as human KC and that AREG silencing can overcome cetuximab resistance in HNSCC cells.
Modeling tumor promoting extracellular matrix dynamics in 3-D suspension culture R Russell, T Webster, M Prisco, D Cognetti, A Luginbuhl, J Curry, A Fertala, J Rosenbloom and A South TJU, Philadelphia, PA Increased tissue stiffness, excessive extracellular matrix (ECM) densification and accumulation of collagen fibers and crosslinks are all associated with tumor cell invasion, immune evasion and tumor progression. We have developed a novel assay that recapitulates collagen fiber formation and tumor fibroblast collagen remodeling in vitro. Our method relies on tissue engineered from primary fibroblasts in suspension, and thus avoids the artificial mechanical stress associated with 2D culture on rigid substrates. Using this assay we measure collagen content and crosslinking via colorimetric analysis of total versus immature collagen fibers, relying upon collagen-specific Sirius red staining and analyses of birefringent collagen fibrillar deposits with the use of a polarizing microscope. Quantification of areas occupied by red-colored and green-colored fibrils is therefore a measure of collagen maturation and crosslinking, with red birefringence an indication of mature collagen fibrils and green birefringence an indication of immature collagen fibrils, as demonstrated by the effects of TGFb1 ligand stimulation and TGFb receptor type I inhibition. An increase of immature collagen fibrils over time is indicative of increased remodeling of collagen-rich ECM. Using multiple patient samples, we determined that collagen assemblies produced by squamous cell carcinoma (SCC) fibroblasts (n¼4) are continually remodeled in contrast to those from isogenic normal fibroblasts (n¼3), and that these differences correlate with invasion when fibroblast matrices are co-cultured with SCC keratinocytes. We are further investigating the parameters of collagen remodeling and tumor cell invasion with the use of b-aminopropionitrile, an irreversible inhibitor of lysyl oxidase, recombinant lysyl oxidase and the lysine analogue trans-4,5-dehydrolysine.
Microbiome controls mast cell differentiation in the skin Z Wang1, N Mascarenhas2, L Eckmann2, Y Miyamoto2, X Sun2, T Kawakami2 and A Di Nardo2 1 University of California, San Diego, La Jolla, CA and 2 University of California San Diego, La Jolla, CA Commensal microbiota play important roles in the skin’s immune system. Originally from bone marrow, mast cell (MC) progenitors enter the skin and become mature in the surrounding microenvironment of skin. Previous studies demonstrate that murine MCs do not fully mature until 8 to 15 days after birth. We hypothesized that skin commensal bacteria and their products have important effects on MC maturation in the skin. Here we show that in skin, germ-free (GF) mice express low levels of stem cell factor (SCF), a critical MC differentiation factor, and their skin MCs are less mature. Immunofluorescence staining and FACS results revealed a smaller population of c-Kit positive MCs in the GF mice that can be normalized after being co-housed with conventional mice for 5 weeks to reconstitute their microbiome. Real time qPCR results of toluidine blue positive cells in the skin, collected by laser capture microdissection, showed that MCs in GF skin expressed lower levels of various MC markers including chymase, tryptase beta 2, MC protease 4, c-Kit, and cathelicidin antimicrobial peptide. Furthermore, compound 48/80, a known degranulation agent, induced less edema in the paws of GF mice than in those of conventional mice. We also found that lipoteichoic acid (a major constituent of the cell wall of Staphylococcus) promotes SCF production in keratinocytes through TLR2, which enhances the development of skin MCs. Our findings are important for the skin diseases involving MCs, such as atopic dermatitis or psoriasis, in which the change in the skin commensal microbiota has been proven to be part of their pathogenesis.
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Proteomic evaluation of desmosomes reveals novel components essential for maintaining epidermal integrity KA Badu-Nkansah1, J Underwood2 and T Lechler3 1 Department of Cell Biology, Duke University School of Medicine, Durham, NC, 2 Department of Dermatology, Duke University School of Medicine, Durham, NC and 3 Duke University, Durham, NC Desmosomes are cell-cell adhesion structures that provide mechanical robustness to the epidermis and heart. Perturbation of desmosomes occurs in genetic, autoimmune and infectious disease and can lead to blistering of the skin. While the core components of the desmosome were identified decades ago, more recent work has identified additional desmosome-associated proteins that have either regulatory and/or non-canonical functions. Here we used proximity biotinylation and quantitative mass spectrometry to identify novel desmosome-associated proteins. We performed separate analyses to enrich for proteins at the outer and inner dense plaques and identified many candidates. Remarkably, we found a significant overlap with proteins associated with adherens junctions. We show that this is due, in part, to promiscuity in the association of some proteins with adherens junctions and desmosomes in different tissues. In addition, we demonstrate that a subset of the identified proteins colocalize with core desmosomal proteins and require these proteins for their localization. Finally, we performed functional evaluation of the role of Crk and Crkl, two homologous proteins that were identified in our analysis. Loss of Crk/Crkl in cultured keratinocytes and in the mouse epidermis resulted in defects in keratin organization, impaired adhesion, and neonatal lethality. These data position Crk/Crkl as crucial regulators of the desmosome:keratin interaction which is essential for mechanical integrity of the skin and highlights the utility of the approach to identify novel regulators of the desmosome.
S96 Journal of Investigative Dermatology (2017), Volume 137
Beneficial effects of Silybum marianum extract and silymarin on regulation of decorin and biglycan M Shin1, J Lim2, H Lee2, J Oh1, Y Kim1, K Kim3, S Cho4 and J Chung2 1 Seoul National University Hospital, Seoul, Seoul-t’ukpyolsi, Republic of Korea, 2 Seoul National University, Seoul, Seoul-t’ukpyolsi, Republic of Korea, 3 Department of Dermatology, Seoul National University College of Medicine, Seoul, Seoul-t’ukpyolsi, Republic of Korea and 4 Seoul National University Boramae Medical Center, Seoul, Seoul-t’ukpyolsi, Republic of Korea Silybum marianum (milk thistle) extract (SME) is well known to have a beneficial effect on improvement of liver health; however, its effect on skin aging has been rarely studied. Major active component of SME is known as silymarin, which contains silibinin, isosilibinin, silicristin, silidianin, and others. In skin aging, loss of collagen and increase of their degrading enzyme, matrix metalloproteinase-1 (MMP-1), and reduction of collagen-supporting proteoglycans, including decorin and biglycan, are notable characteristics. Therefore, in this study, we investigated the effects of SME on the matrix protein regulation in primary human dermal fibroblasts (HDFs). Treatment with SME increased expressions of procollagen, decorin, and biglycan in HDFs, while reduced MMP-1 expression. Furthermore, molecular sizes of decorin and biglycan were also elevated, suggesting that longer dermatan sulfate chain synthesis was induced by treatment with SME. Treatment with silymarin also showed similar results in HDFs. In addition, we also found that both SME and silymarin have direct inhibitory effects on granzyme B and neutrophil elastase, which are enzymes degrading proteoglycans, using recombinant human biglycan and decorin. Degradation of recombinant human biglycan and decorin by recombinant human granzyme B or neutrophil elastase were blocked by addition of SME or silymarin. Therefore, with these benficial effects on biglycan and decorin regualtion, SME and silymarin can be new candidates for anti-aging molecules.
555
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557
Dynamic organization and order of desmoglein 3 in desmosomes determined by fluorescence polarization microscopy EI Bartle, TM Urner and AL Mattheyses Emory University School of Medicine, Atlanta, GA Desmosomes are macromolecular cell-cell complexes responsible for providing strong adhesion between epidermal keratinocytes by coupling extracellular adhesions to the intracellular intermediate filament cytoskeleton. Electron microscopy studies have revealed that the cadherin proteins in the adhesive core of desmosomes appear to be organized.However,due to the size and molecular complexity of desmosomes, how cadherin order relates to adhesion in living cells has been challenging to study. We addressed this challenge by developing and applying fluorescence polarization microscopy to study protein order in desmosomes. Fluorescence polarization microscopy allows characterization of collective protein order or disorder within macromolecular complexes. The cadherin desmoglein 3 was tagged with GFP (Dsg3-DEA-GFP) and transfected into HaCaT human keratinocyte cells. Imaging with fluorescence polarization microscopy revealed that the extracellular domain of desmoglein 3 is ordered in desmosomes in living cells. To investigate if this order is correlated to adhesive function, calcium was depleted from the media of HaCaT cells with calcium sensitive desmosomes. This led to a progressive loss of adhesive strength over 30 minutes, as determined by a cell fragmentation assay. Time courses of individual cells imaged with fluorescence polarization revealed a concurrent loss of Dsg3-DEA-GFP order. Our results establish that the order and organization of the desmosomal cadherins can be measured in living cells. We propose that extracellular domain order can be used as a optical biomarkerallowing spatio-temporal measurement of adhesive strength in individual desmosomes. These results demonstrate that fluorescence polarization microscopy is a novel approach to study desmosome organization and function in living cells and in human disease.
Amphiregulin silencing overcomes resistance to the EGF receptor inhibitor cetuximab in head and neck squamous cell carcinoma SW Stoll1 and J Elder2 1 University of Michigan, Ann Arbor, MI and 2 Dept. of Dermatology, U.of Michigan, Ann Arbor, MI EGF receptor (EGFR) is central to epithelial cell physiology and its deregulated signaling has been implicated in the pathogenesis of various epithelial neoplasias. Overexpression of EGFR is an important prognostic factor in head and neck squamous cell carcinoma (HNSCC) and EGFR blockade has emerged as a significant yet imperfect treatment modality for these tumors. In human skin keratinocytes (KC) we have previously shown that interfering with EGFR signaling through silencing of the EGFR ligand amphiregulin (AREG) leads to a pronounced state of G2/M/cytokinesis growth arrest that cannot be reversed by addition of exogenous EGFR ligands, including AREG itself. To compare the efficacy of AREG silencing and EGFR blockade in human skin KC and malignant epithelial cells we treated the HNSCC cell lines UMSCC-22A and UMSCC-22B, which are derived from a primary HNSCC and its metastasis, with the EGFR inhibitor cetuximab. Incubation of these cells with various concentrations of cetuximab led to a reduction in cell numbers which was much more modest than in immortalized but non-transformed human N/TERT-KC (UMSCC-22A: 45.2% of control at 500 ng/ml and 42.5% at 1000 ng/ml; UMSCC-22B: 46.9% and 35.4%; N/TERT: 18.1% and 11.1%; n¼2-4). In contrast, and very similar to N/TERT-KC (JID 136:444), shRNA-mediated AREG silencing reduced HNSCC cell growth by more than 88% (UMSCC-22A: 12 +/- 2.3% of control; UMSCC-22B 11.5 +/- 1.1%, mean +/- SEM, n¼4). Because of the already strong inhibition of cell growth in response to AREG silencing, a combination of cetuximab and AREG shRNA resulted only in a slight further improvement. Similar to N/TERT-KC, AREG silencing in these HNSCC cell lines led to the appearance of numerous bi- and multinucleated cells, many of which appeared to be undergoing mitotic catastrophe. Together, our findings suggest that UMSCC-22A and UMSCC-22B cells are similarly sensitive to AREG silencing as human KC and that AREG silencing can overcome cetuximab resistance in HNSCC cells.
Modeling tumor promoting extracellular matrix dynamics in 3-D suspension culture R Russell, T Webster, M Prisco, D Cognetti, A Luginbuhl, J Curry, A Fertala, J Rosenbloom and A South TJU, Philadelphia, PA Increased tissue stiffness, excessive extracellular matrix (ECM) densification and accumulation of collagen fibers and crosslinks are all associated with tumor cell invasion, immune evasion and tumor progression. We have developed a novel assay that recapitulates collagen fiber formation and tumor fibroblast collagen remodeling in vitro. Our method relies on tissue engineered from primary fibroblasts in suspension, and thus avoids the artificial mechanical stress associated with 2D culture on rigid substrates. Using this assay we measure collagen content and crosslinking via colorimetric analysis of total versus immature collagen fibers, relying upon collagen-specific Sirius red staining and analyses of birefringent collagen fibrillar deposits with the use of a polarizing microscope. Quantification of areas occupied by red-colored and green-colored fibrils is therefore a measure of collagen maturation and crosslinking, with red birefringence an indication of mature collagen fibrils and green birefringence an indication of immature collagen fibrils, as demonstrated by the effects of TGFb1 ligand stimulation and TGFb receptor type I inhibition. An increase of immature collagen fibrils over time is indicative of increased remodeling of collagen-rich ECM. Using multiple patient samples, we determined that collagen assemblies produced by squamous cell carcinoma (SCC) fibroblasts (n¼4) are continually remodeled in contrast to those from isogenic normal fibroblasts (n¼3), and that these differences correlate with invasion when fibroblast matrices are co-cultured with SCC keratinocytes. We are further investigating the parameters of collagen remodeling and tumor cell invasion with the use of b-aminopropionitrile, an irreversible inhibitor of lysyl oxidase, recombinant lysyl oxidase and the lysine analogue trans-4,5-dehydrolysine.
Microbiome controls mast cell differentiation in the skin Z Wang1, N Mascarenhas2, L Eckmann2, Y Miyamoto2, X Sun2, T Kawakami2 and A Di Nardo2 1 University of California, San Diego, La Jolla, CA and 2 University of California San Diego, La Jolla, CA Commensal microbiota play important roles in the skin’s immune system. Originally from bone marrow, mast cell (MC) progenitors enter the skin and become mature in the surrounding microenvironment of skin. Previous studies demonstrate that murine MCs do not fully mature until 8 to 15 days after birth. We hypothesized that skin commensal bacteria and their products have important effects on MC maturation in the skin. Here we show that in skin, germ-free (GF) mice express low levels of stem cell factor (SCF), a critical MC differentiation factor, and their skin MCs are less mature. Immunofluorescence staining and FACS results revealed a smaller population of c-Kit positive MCs in the GF mice that can be normalized after being co-housed with conventional mice for 5 weeks to reconstitute their microbiome. Real time qPCR results of toluidine blue positive cells in the skin, collected by laser capture microdissection, showed that MCs in GF skin expressed lower levels of various MC markers including chymase, tryptase beta 2, MC protease 4, c-Kit, and cathelicidin antimicrobial peptide. Furthermore, compound 48/80, a known degranulation agent, induced less edema in the paws of GF mice than in those of conventional mice. We also found that lipoteichoic acid (a major constituent of the cell wall of Staphylococcus) promotes SCF production in keratinocytes through TLR2, which enhances the development of skin MCs. Our findings are important for the skin diseases involving MCs, such as atopic dermatitis or psoriasis, in which the change in the skin commensal microbiota has been proven to be part of their pathogenesis.
558
559
Proteomic evaluation of desmosomes reveals novel components essential for maintaining epidermal integrity KA Badu-Nkansah1, J Underwood2 and T Lechler3 1 Department of Cell Biology, Duke University School of Medicine, Durham, NC, 2 Department of Dermatology, Duke University School of Medicine, Durham, NC and 3 Duke University, Durham, NC Desmosomes are cell-cell adhesion structures that provide mechanical robustness to the epidermis and heart. Perturbation of desmosomes occurs in genetic, autoimmune and infectious disease and can lead to blistering of the skin. While the core components of the desmosome were identified decades ago, more recent work has identified additional desmosome-associated proteins that have either regulatory and/or non-canonical functions. Here we used proximity biotinylation and quantitative mass spectrometry to identify novel desmosome-associated proteins. We performed separate analyses to enrich for proteins at the outer and inner dense plaques and identified many candidates. Remarkably, we found a significant overlap with proteins associated with adherens junctions. We show that this is due, in part, to promiscuity in the association of some proteins with adherens junctions and desmosomes in different tissues. In addition, we demonstrate that a subset of the identified proteins colocalize with core desmosomal proteins and require these proteins for their localization. Finally, we performed functional evaluation of the role of Crk and Crkl, two homologous proteins that were identified in our analysis. Loss of Crk/Crkl in cultured keratinocytes and in the mouse epidermis resulted in defects in keratin organization, impaired adhesion, and neonatal lethality. These data position Crk/Crkl as crucial regulators of the desmosome:keratin interaction which is essential for mechanical integrity of the skin and highlights the utility of the approach to identify novel regulators of the desmosome.
S96 Journal of Investigative Dermatology (2017), Volume 137
Beneficial effects of Silybum marianum extract and silymarin on regulation of decorin and biglycan M Shin1, J Lim2, H Lee2, J Oh1, Y Kim1, K Kim3, S Cho4 and J Chung2 1 Seoul National University Hospital, Seoul, Seoul-t’ukpyolsi, Republic of Korea, 2 Seoul National University, Seoul, Seoul-t’ukpyolsi, Republic of Korea, 3 Department of Dermatology, Seoul National University College of Medicine, Seoul, Seoul-t’ukpyolsi, Republic of Korea and 4 Seoul National University Boramae Medical Center, Seoul, Seoul-t’ukpyolsi, Republic of Korea Silybum marianum (milk thistle) extract (SME) is well known to have a beneficial effect on improvement of liver health; however, its effect on skin aging has been rarely studied. Major active component of SME is known as silymarin, which contains silibinin, isosilibinin, silicristin, silidianin, and others. In skin aging, loss of collagen and increase of their degrading enzyme, matrix metalloproteinase-1 (MMP-1), and reduction of collagen-supporting proteoglycans, including decorin and biglycan, are notable characteristics. Therefore, in this study, we investigated the effects of SME on the matrix protein regulation in primary human dermal fibroblasts (HDFs). Treatment with SME increased expressions of procollagen, decorin, and biglycan in HDFs, while reduced MMP-1 expression. Furthermore, molecular sizes of decorin and biglycan were also elevated, suggesting that longer dermatan sulfate chain synthesis was induced by treatment with SME. Treatment with silymarin also showed similar results in HDFs. In addition, we also found that both SME and silymarin have direct inhibitory effects on granzyme B and neutrophil elastase, which are enzymes degrading proteoglycans, using recombinant human biglycan and decorin. Degradation of recombinant human biglycan and decorin by recombinant human granzyme B or neutrophil elastase were blocked by addition of SME or silymarin. Therefore, with these benficial effects on biglycan and decorin regualtion, SME and silymarin can be new candidates for anti-aging molecules.