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447 Members of the lysyl oxidase family of enzymes (LOX, LOX-L1, LOX-L2) may play a role in keloid pathogenesis
Growth Factors, Cell Adhesion & Matrix Biology | ABSTRACTS 445
446
Discovery of an alternate, flotillin-dependent, clathrin-mediated endocytosis pathway for IGF-1 receptor internalization and signaling in keratinocytes DM Dam, S Jelsma and AS Paller Dermatology, Northwestern University, Chicago, IL Suppressed insulin/insulin-like growth factor-1 (IGF-1) receptor signaling (“insulin resistance”) in type 2 diabetes leads to impaired wound re-epithelialization. In normal human epidermal keratinocytes (NHEKs), this pathway is primarily activated through the IGF-1 receptor (IGF1R). Despite the critical role of IGF1R in migration and re-epithelialization, the mechanism of its endocytosis and subsequent activation in NHEKs is poorly understood. Flotillin (flot-1), a noncaveolar lipid raft marker, affects receptor tyrosine kinase signaling in cancer cells, but litle is known about its role in normal cells. In starved NHEKs, we found surface IGF1R colocalized with flot-1 using microscopy and coimmunoprecipitation (coIP). Upon IGF-1 stimulation, IGF1R colocalizes both with flot-1 in lipid rafts and with the adaptin complex (aAP1/2) and clathrin in clathrin pits. Interestingly, IGF-1 also promotes colocalization and coIP of flot-1 with clathrin. Despite blockade of endocytosis via clathrin pits with chlorpromazine (CPMZ) or shRNA knockdown of aAP1/2, IGF1R still endocytoses and associates with both flot-1 and clathrin. In addition, flot-1 knockdown or dissociation of flot-1 from IGF1R and clathrin after lipid raft dissolution using methyl-b-cyclodextrin (MbCD) fails to prevent IGF1R internalization with clathrin. In contrast, concurrent shRNA knockdown of flot-1 and aAP1/2 eliminates the association of IGF1R with clathrin and prevents endocytosis and signaling. Similarly, treatment of IGF-1-stimulated NHEKs with both CPMZ and MbCD fully blocks endocytosis and completely abolishes IGF-1-induced phosphorylation of IGF1R, Akt and MEK. These data suggest that IGF1R endocytosis and signaling are controlled by clathrin-dependent endocytosis, mediated by one of two adaptors: flotillin in noncaveolar lipid rafts or the aAP1/2 complex in clathrin pits. The discovery of a flotillin-dependent, clathrin-mediated endocytosis pathway for IGF1R provides a new avenue for drug discovery for diseases with aberrant regulation of IGF1R signaling.
Keloid fibroblast exosomes alter gene expression profile in normal fibroblasts J Andrews1, L Liu1, E Macarak1,2, J Rosenbloom1,2 and J Uitto1 1 Dermatology, Thomas Jefferson University, Philadelphia, PA and 2 The Joan and Joel Rosenbloom Center for Fibrotic Diseases, Thomas Jefferson University, Philadelphia, PA Keloids represent benign, fibroproliferative tumors arising in response to dermal injury. Exosomes are small (30-200 nm) secretory vesicles that contain a myriad of proteins, mRNA and DNA, and are secreted from most cell types. In the present study, we demonstrate the utility of using keloid fibroblast-derived exosomes to alter the gene expression profile of normal dermal fibroblasts. Keloid tissue and normal skin were obtained following surgical cosmetic excision or panniculectomy. Tissue explant cultures were grown under sterile conditions and keloid fibroblasts (KF) were grown to confluence in exosome-depleted culture media. Following a 48-hour incubation, the culture medium was removed, and secreted exosomes were isolated with ExoQuick solution according to the manufacturer’s protocol. Utilizing NanoSight image analysis, the average particle size was 133.5 ( 18.1) nm with a concentration of 2.52 x 108 particles/ml in the exosome isolate. Total protein of the keloid exosome was determined to be 263.8 mg/ml. Normal fibroblasts at passage 4 were then incubated in exosome-depleted media either with or without 50 mg of keloid exosome for a 24 hour period. After the 24 hr incubation, normal fibroblasts were lysed, total RNA extracted and reverse transcribed into cDNA. RT-qPCR was performed to yield comparative gene expression profiles in both cell cultures with TGF-b (1.3), Col-1 (1.1), LOX (1.7), and LOX-L1 (1.8 fold), all demonstrating up-regulation in cells treated with keloid exosomes compared to untreated cultures. The keloid exosome represents an additional compartment through which cell-cell communication takes place and which can perpetuate pathologic fibrotic signaling. Its presence may explain, in part, the extensive growth and matrix deposition characteristic of keloids.
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Members of the lysyl oxidase family of enzymes (LOX, LOX-L1, LOX-L2) may play a role in keloid pathogenesis J Andrews1, L Liu1, E Macarak1,2, J Rosenbloom1,2 and J Uitto1 1 Dermatology, Thomas Jefferson University, Philadelphia, PA and 2 The Joan and Joel Rosenbloom Center for Fibrotic Diseases, Thomas Jefferson University, Philadelphia, PA Keloids are benign fibrous tumors composed predominantly of thick, haphazardly arranged type I collagen. LOX is a copper-dependent amine oxidase that catalyzes the formation of collagen-collagen crosslinking through oxidative deamination of lysine residues. It belongs to a family of LOX-like enzymes, including LOX-L1 and L2. In the present study we demonstrate the up-regulation of LOX, L1 and L2 in keloid tissue compared to control skin, and maintenance of their higher expression in two separate mouse models. Keloid tissue and normal skin were obtained following surgical cosmetic excision or panniculectomy. Total RNA was extracted from 40 mg tissue pieces and reverse transcribed. cDNA products were utilized for RT-qPCR analysis, yielding a baseline gene expression. As a result, LOX (3.3), L1 (16.1), and L2 (11.2) together with TGF-b (2.5 fold) were all up-regulated in keloid tissue. Next, tissue explant cultures were made under sterile conditions for both keloid and normal skin samples. Confluent fibroblasts at passages 4-6 were dynamically seeded onto polylactic acid scaffolds for 7 days and then subcutaneously implanted for 2 weeks into Rag1 mice. RT-qPCR analysis of extracted scaffolds yielded similar up-regulation of TGF-b (1.4), LOX (10.4), L1 (5.6), and L2 (2.1 fold). A separate model of tissue fibrosis utilizing daily 100 mg/ml subcutaneous bleomycin injections into C3H mice for a total of two weeks also displayed up-regulation of TGF-b (1.4), L1 (2.9), and L2 (3.8) but not LOX. Dermal thickness was measured to be approximately 55% larger in the bleomycin group than controls. BAPN, an inhibitor of LOX, was then injected on a bi-weekly basis, concurrently with bleomycin for 2 weeks, and dermal thickness was shown not to be different from controls. These experiments imply the importance of LOX, L1, and L2 in keloid pathogenesis and the utility of BAPN as a potential therapeutic agent.
Sub-toxic hydrogen peroxide exposure significantly reduces expression of extracellular matrix and dermal epidermal junction components in 3-D skin tissue, mimicking key features of aging skin S Chon1, L Piccotti1, D Ngai2 and K Rodriguez1 1 Skin and vaginal physiology, KimberlyClark Corporation, Roswell, GA and 2 Skin and vaginal physiology, Kimberly-Clark Corporation, Neenah, WI Oxidative stress has been defined as a primary cause of skin aging. It can be induced extrinsically via prolonged exposure to environmental aggressors such as solar UV and intrinsically due to reduced cellular functions of detoxification, antioxidant defenses, mitochondrial respiration and DNA repair process during aging. This study aimed to develop a three-dimensional tissue culture model of aging skin by exposing commercially available human skin equivalents to hydrogen peroxide stimulus. Tissues were treated with 2- 10mM of hydrogen peroxide by either a single acute exposure or multiple chronic exposures. Tissue histology and cytotoxicity were examined by Hematoxylin and eosin staining and Lactate dehydrogenase assays, respectively. Gene and protein expression of extracellular matrix (ECM) and dermal epidermal junction (DEJ) components were analyzed by QPCR and multiplex assays. Among all conditions tested, 2mM hydrogen peroxide treatment for 3-6 days significantly reduced expression of multiple ECM and DEJ components without detrimental cytoxicity to the tissues. This treatment markedly down-regulated gene expression of Collagens (COL1A1, COL4A1), Elastin, TIMP1 and Tenascin C, coupled with a drastic increase in MMP1 gene expression. Similar changes were observed in protein expression of these markers, confirming results from gene expression analyses. The results demonstrated that oxidative stress induced by 2mM hydrogen peroxide significantly compromised expression of dermal ECM and DEJ components. The identified culture condition mimicked key features of aged skin and can be further utilized as a basic and applied research tool to promote aging skin health.
www.jidonline.org S79
446
Discovery of an alternate, flotillin-dependent, clathrin-mediated endocytosis pathway for IGF-1 receptor internalization and signaling in keratinocytes DM Dam, S Jelsma and AS Paller Dermatology, Northwestern University, Chicago, IL Suppressed insulin/insulin-like growth factor-1 (IGF-1) receptor signaling (“insulin resistance”) in type 2 diabetes leads to impaired wound re-epithelialization. In normal human epidermal keratinocytes (NHEKs), this pathway is primarily activated through the IGF-1 receptor (IGF1R). Despite the critical role of IGF1R in migration and re-epithelialization, the mechanism of its endocytosis and subsequent activation in NHEKs is poorly understood. Flotillin (flot-1), a noncaveolar lipid raft marker, affects receptor tyrosine kinase signaling in cancer cells, but litle is known about its role in normal cells. In starved NHEKs, we found surface IGF1R colocalized with flot-1 using microscopy and coimmunoprecipitation (coIP). Upon IGF-1 stimulation, IGF1R colocalizes both with flot-1 in lipid rafts and with the adaptin complex (aAP1/2) and clathrin in clathrin pits. Interestingly, IGF-1 also promotes colocalization and coIP of flot-1 with clathrin. Despite blockade of endocytosis via clathrin pits with chlorpromazine (CPMZ) or shRNA knockdown of aAP1/2, IGF1R still endocytoses and associates with both flot-1 and clathrin. In addition, flot-1 knockdown or dissociation of flot-1 from IGF1R and clathrin after lipid raft dissolution using methyl-b-cyclodextrin (MbCD) fails to prevent IGF1R internalization with clathrin. In contrast, concurrent shRNA knockdown of flot-1 and aAP1/2 eliminates the association of IGF1R with clathrin and prevents endocytosis and signaling. Similarly, treatment of IGF-1-stimulated NHEKs with both CPMZ and MbCD fully blocks endocytosis and completely abolishes IGF-1-induced phosphorylation of IGF1R, Akt and MEK. These data suggest that IGF1R endocytosis and signaling are controlled by clathrin-dependent endocytosis, mediated by one of two adaptors: flotillin in noncaveolar lipid rafts or the aAP1/2 complex in clathrin pits. The discovery of a flotillin-dependent, clathrin-mediated endocytosis pathway for IGF1R provides a new avenue for drug discovery for diseases with aberrant regulation of IGF1R signaling.
Keloid fibroblast exosomes alter gene expression profile in normal fibroblasts J Andrews1, L Liu1, E Macarak1,2, J Rosenbloom1,2 and J Uitto1 1 Dermatology, Thomas Jefferson University, Philadelphia, PA and 2 The Joan and Joel Rosenbloom Center for Fibrotic Diseases, Thomas Jefferson University, Philadelphia, PA Keloids represent benign, fibroproliferative tumors arising in response to dermal injury. Exosomes are small (30-200 nm) secretory vesicles that contain a myriad of proteins, mRNA and DNA, and are secreted from most cell types. In the present study, we demonstrate the utility of using keloid fibroblast-derived exosomes to alter the gene expression profile of normal dermal fibroblasts. Keloid tissue and normal skin were obtained following surgical cosmetic excision or panniculectomy. Tissue explant cultures were grown under sterile conditions and keloid fibroblasts (KF) were grown to confluence in exosome-depleted culture media. Following a 48-hour incubation, the culture medium was removed, and secreted exosomes were isolated with ExoQuick solution according to the manufacturer’s protocol. Utilizing NanoSight image analysis, the average particle size was 133.5 ( 18.1) nm with a concentration of 2.52 x 108 particles/ml in the exosome isolate. Total protein of the keloid exosome was determined to be 263.8 mg/ml. Normal fibroblasts at passage 4 were then incubated in exosome-depleted media either with or without 50 mg of keloid exosome for a 24 hour period. After the 24 hr incubation, normal fibroblasts were lysed, total RNA extracted and reverse transcribed into cDNA. RT-qPCR was performed to yield comparative gene expression profiles in both cell cultures with TGF-b (1.3), Col-1 (1.1), LOX (1.7), and LOX-L1 (1.8 fold), all demonstrating up-regulation in cells treated with keloid exosomes compared to untreated cultures. The keloid exosome represents an additional compartment through which cell-cell communication takes place and which can perpetuate pathologic fibrotic signaling. Its presence may explain, in part, the extensive growth and matrix deposition characteristic of keloids.
447
448
Members of the lysyl oxidase family of enzymes (LOX, LOX-L1, LOX-L2) may play a role in keloid pathogenesis J Andrews1, L Liu1, E Macarak1,2, J Rosenbloom1,2 and J Uitto1 1 Dermatology, Thomas Jefferson University, Philadelphia, PA and 2 The Joan and Joel Rosenbloom Center for Fibrotic Diseases, Thomas Jefferson University, Philadelphia, PA Keloids are benign fibrous tumors composed predominantly of thick, haphazardly arranged type I collagen. LOX is a copper-dependent amine oxidase that catalyzes the formation of collagen-collagen crosslinking through oxidative deamination of lysine residues. It belongs to a family of LOX-like enzymes, including LOX-L1 and L2. In the present study we demonstrate the up-regulation of LOX, L1 and L2 in keloid tissue compared to control skin, and maintenance of their higher expression in two separate mouse models. Keloid tissue and normal skin were obtained following surgical cosmetic excision or panniculectomy. Total RNA was extracted from 40 mg tissue pieces and reverse transcribed. cDNA products were utilized for RT-qPCR analysis, yielding a baseline gene expression. As a result, LOX (3.3), L1 (16.1), and L2 (11.2) together with TGF-b (2.5 fold) were all up-regulated in keloid tissue. Next, tissue explant cultures were made under sterile conditions for both keloid and normal skin samples. Confluent fibroblasts at passages 4-6 were dynamically seeded onto polylactic acid scaffolds for 7 days and then subcutaneously implanted for 2 weeks into Rag1 mice. RT-qPCR analysis of extracted scaffolds yielded similar up-regulation of TGF-b (1.4), LOX (10.4), L1 (5.6), and L2 (2.1 fold). A separate model of tissue fibrosis utilizing daily 100 mg/ml subcutaneous bleomycin injections into C3H mice for a total of two weeks also displayed up-regulation of TGF-b (1.4), L1 (2.9), and L2 (3.8) but not LOX. Dermal thickness was measured to be approximately 55% larger in the bleomycin group than controls. BAPN, an inhibitor of LOX, was then injected on a bi-weekly basis, concurrently with bleomycin for 2 weeks, and dermal thickness was shown not to be different from controls. These experiments imply the importance of LOX, L1, and L2 in keloid pathogenesis and the utility of BAPN as a potential therapeutic agent.
Sub-toxic hydrogen peroxide exposure significantly reduces expression of extracellular matrix and dermal epidermal junction components in 3-D skin tissue, mimicking key features of aging skin S Chon1, L Piccotti1, D Ngai2 and K Rodriguez1 1 Skin and vaginal physiology, KimberlyClark Corporation, Roswell, GA and 2 Skin and vaginal physiology, Kimberly-Clark Corporation, Neenah, WI Oxidative stress has been defined as a primary cause of skin aging. It can be induced extrinsically via prolonged exposure to environmental aggressors such as solar UV and intrinsically due to reduced cellular functions of detoxification, antioxidant defenses, mitochondrial respiration and DNA repair process during aging. This study aimed to develop a three-dimensional tissue culture model of aging skin by exposing commercially available human skin equivalents to hydrogen peroxide stimulus. Tissues were treated with 2- 10mM of hydrogen peroxide by either a single acute exposure or multiple chronic exposures. Tissue histology and cytotoxicity were examined by Hematoxylin and eosin staining and Lactate dehydrogenase assays, respectively. Gene and protein expression of extracellular matrix (ECM) and dermal epidermal junction (DEJ) components were analyzed by QPCR and multiplex assays. Among all conditions tested, 2mM hydrogen peroxide treatment for 3-6 days significantly reduced expression of multiple ECM and DEJ components without detrimental cytoxicity to the tissues. This treatment markedly down-regulated gene expression of Collagens (COL1A1, COL4A1), Elastin, TIMP1 and Tenascin C, coupled with a drastic increase in MMP1 gene expression. Similar changes were observed in protein expression of these markers, confirming results from gene expression analyses. The results demonstrated that oxidative stress induced by 2mM hydrogen peroxide significantly compromised expression of dermal ECM and DEJ components. The identified culture condition mimicked key features of aged skin and can be further utilized as a basic and applied research tool to promote aging skin health.
www.jidonline.org S79