Presentation is loading. Please wait.

Presentation is loading. Please wait.

Novel Functions of Intracellular IL-1ra in Human Dermal Fibroblasts: Implications in the Pathogenesis of Fibrosis  Siva Kanangat, Arnold E. Postlethwaite,

Published byImogen O’Connor’ Modified over 5 years ago

Similar presentations

Presentation on theme: "Novel Functions of Intracellular IL-1ra in Human Dermal Fibroblasts: Implications in the Pathogenesis of Fibrosis  Siva Kanangat, Arnold E. Postlethwaite,"— Presentation transcript:

1 Novel Functions of Intracellular IL-1ra in Human Dermal Fibroblasts: Implications in the Pathogenesis of Fibrosis  Siva Kanangat, Arnold E. Postlethwaite, Gloria C. Higgins, Karen A. Hasty  Journal of Investigative Dermatology  Volume 126, Issue 4, Pages (April 2006) DOI: /sj.jid Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Overexpression of icIL-1ra in HF-icIL-1ra. (a) Equal number of cells (HF-icIL-1ra and HF-Vector) were stimulated with 0 and 1.0ng of hrIL-1β or 10ng/ml of hrTNF-α. After 24hours, total RNA was extracted and mRNA levels of icIL-1ra and GAPDH were estimated by real-time RT-PCR. Ratios of icIL-1ra type 1 to GAPDH messages are plotted in the graph. (b) Equal numbers of HF-icIL-1ra and HF-Vector maintained in complete DMEM for 48hours were harvested and lysed. The clarified cell lysates were tested for icIL-1ra type 1 by ELISA. The error bars indicate mean±SD of three separate experiments on the same batch of stably transfected fibroblasts. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Myofibroblast morphology of HF-icIL-1ra. (a) HF-Vector with normal spindle-shaped fibroblast morphology after culturing in complete DMEM for 6 weeks with medium changes every 5 days. (b) HF-icIL-1ra with myofibroblast-like morphology. (c, d) Cells were fixed and permeabilized (Cytofix/Cytoperm) and were subjected to immunoperoxidase staining with mouse anti-human α-SMA (Sigma) clone 1A 4 monoclonal antibody followed by color reaction developed by a streptavidin–horseradish peroxidase system as described. (c) HF-Vector with little α-SMA staining. (d) HF-icIL-1ra with α-SMA staining. (e) HFF-TGF-passed cells with myofibroblast-like morphology. (f) HFF-TGF with intense staining for α-SMA. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Enhanced levels of α-SMA and PAI mRNA in HF- icIL-1ra. HF-icIL-1ra and HF-Vector were maintained in complete DMEM for 6 weeks with medium changes every 5 days. Cells were harvested and total cellular RNA was extracted and reverse transcribed. The cDNA thus obtained was amplified by real-time PCR and quantified by SYBR Green using α-SMA primers and PAI primers (Table 4). The values are expressed as ratios of Ct values of α-SMA to those of the housekeeping gene GAPDH. The values indicate mean±SD of three independent experiments performed on the same batch of stably transfected fibroblasts. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Reduced mRNA in HF-icIL-1ra. HF-icIL-1ra and HF-Vector were maintained in complete DMEM for 6 weeks with medium changes every 5 days. Cells were harvested in Tri-Reagent and total cellular RNA was extracted. One microgram of total RNA was reverse transcribed in a 20μl reverse transcription reaction mixture and 5.0μl of the cDNA was amplified by 28 cycles of PCR using primers specific for GAPDH and MMP-1. The PCR products were analyzed on a 2% agarose gel, stained with ethidium bromide, and photographed. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Reduced expression of IL-1- and TNF-induced MMP- 1mRNA in HF-icIL-1ra. HF-icIL-1ra and HF-Vector were stimulated with 1.0ng/ml IL-1β or10ng/ml TNF-α for 12–16hours. MMP-1 and GAPDH message levels were estimated using real-time RT-PCR. Three separate experiments were performed on the same batch of stably transfected fibroblasts. The results are represented as the reciprocal of the ratios of the Ct values of MMP-1 to those of GAPDH (the housekeeping gene). Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Reduced levels of MMP-1 protein in HF-icIL-1ra type 1. HF-icIL-1ra and HF-Vector were cultured for 48hours with hrIL-1β (1.0ng/ml) or hrTNF-α (5ng/ml) and MMP-1 protein secreted into the culture medium was measured by ELISA. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 Normal expression of TIMP-1 mRNA in fibroblasts overexpressing icIL-1ra. HF-icIL-1ra type 1 HF-icIL-1ra and HF-Vector were stimulated with 0 or 10ng/ml TNF-α for 12–16hours. Total cellular RNA was extracted and TIMP-1 and GAPDH message levels were estimated using SYBR Green real-time RT-PCR. The values are expressed as the reciprocal ratios of Ct values of TIMP-1 mRNA and collagen type I mRNA to those of the housekeeping gene GAPDH. The values indicate mean±SD of three independent experiments performed on the same batch of stably transfected fibroblasts. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

9 Figure 8 Transfection of HF-icIL-1ra with antisense oligonucleotide directed toward icIL-1ra restores IL-1-induced MMP-1 mRNA expression. Phosphorothioate-derivatized antisense oligodeoxynucleotide complimentary to −6 to +12 of the natural icIL-1ra was synthesized and oligonucleotide with a scrambled sequence was prepared by a similar method as a control. HF- icIL-1ra stimulated with 1.0ng/ml IL-1β was transfected with 300mM of antisense icIL-1ra type 1 oligonucleotide (24hours prior to stimulation with 1.0ng/ml of IL-1β) using LipofectAMINE. PBS and LipofectAMINE alone served as additional controls. The cells were harvested 12–18hours after stimulation for RNA extraction. Total RNA was reverse transcribed and MMP-1 mRNA was estimated by real-time RT-PCR. The values indicate mean±SD of three independent experiments performed on the same batch of transfected geneticin-selected fibroblasts (PBS: PBS alone; LIPO: LipofectAMINE alone; ANTIS-LIP: antisense icIL-1ra oligonucleotide+LipofectAMINE; SCRAM-LIP: scrambled oligonucleotide+LipofectAMINE). Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Download ppt "Novel Functions of Intracellular IL-1ra in Human Dermal Fibroblasts: Implications in the Pathogenesis of Fibrosis  Siva Kanangat, Arnold E. Postlethwaite,"

Similar presentations

Dkk1-induced inhibition of Wnt signaling in osteoblast differentiation is an underlying mechanism of bone loss in multiple myeloma  Ya-Wei Qiang, Bart.

Dkk1-induced inhibition of Wnt signaling in osteoblast differentiation is an underlying mechanism of bone loss in multiple myeloma  Ya-Wei Qiang, Bart.
IL-18 Downregulates Collagen Production in Human Dermal Fibroblasts via the ERK Pathway  Hee Jung Kim, Seok Bean Song, Jung Min Choi, Kyung Moon Kim,

IL-18 Downregulates Collagen Production in Human Dermal Fibroblasts via the ERK Pathway  Hee Jung Kim, Seok Bean Song, Jung Min Choi, Kyung Moon Kim,
Volume 61, Issue 2, Pages (February 2002)

Volume 61, Issue 2, Pages (February 2002)
NF-κB Accumulation Associated with COL1A1 Transactivators Defects during Chronological Aging Represses Type I Collagen Expression through a –112/–61-bp.

NF-κB Accumulation Associated with COL1A1 Transactivators Defects during Chronological Aging Represses Type I Collagen Expression through a –112/–61-bp.
Differential Regulation by IL-1β and EGF of Expression of Three Different Hyaluronan Synthases in Oral Mucosal Epithelial Cells and Fibroblasts and Dermal.

Differential Regulation by IL-1β and EGF of Expression of Three Different Hyaluronan Synthases in Oral Mucosal Epithelial Cells and Fibroblasts and Dermal.
Exogenous Smad3 Accelerates Wound Healing in a Rabbit Dermal Ulcer Model  Koji Sumiyoshi, Atsuhito Nakao, Yasuhiro Setoguchi, Ko Okumura, Hideoki Ogawa 

Exogenous Smad3 Accelerates Wound Healing in a Rabbit Dermal Ulcer Model  Koji Sumiyoshi, Atsuhito Nakao, Yasuhiro Setoguchi, Ko Okumura, Hideoki Ogawa 
Myocardin-Related Transcription Factors A and B Are Key Regulators of TGF-β1- Induced Fibroblast to Myofibroblast Differentiation  Beverly J. Crider, George.

Myocardin-Related Transcription Factors A and B Are Key Regulators of TGF-β1- Induced Fibroblast to Myofibroblast Differentiation  Beverly J. Crider, George.
Novel Splice Variants of IL-33: Differential Expression in Normal and Transformed Cells  Hidetoshi Tsuda, Mayumi Komine, Masaru Karakawa, Takafumi Etoh,

Novel Splice Variants of IL-33: Differential Expression in Normal and Transformed Cells  Hidetoshi Tsuda, Mayumi Komine, Masaru Karakawa, Takafumi Etoh,
Staphylococcus aureus Stimulates Neutrophil Targeting Chemokine Expression in Keratinocytes through an Autocrine IL-1α Signaling Loop  Florina Olaru,

Staphylococcus aureus Stimulates Neutrophil Targeting Chemokine Expression in Keratinocytes through an Autocrine IL-1α Signaling Loop  Florina Olaru,
Lei Yin, Akimichi Morita, Takuo Tsuji 

Lei Yin, Akimichi Morita, Takuo Tsuji 
NF-κBp65-specific siRNA inhibits expression of genes of COX-2, NOS-2 and MMP-9 in rat IL-1β-induced and TNF-α-induced chondrocytes  Dr C. Lianxu, Ph.D.,

NF-κBp65-specific siRNA inhibits expression of genes of COX-2, NOS-2 and MMP-9 in rat IL-1β-induced and TNF-α-induced chondrocytes  Dr C. Lianxu, Ph.D.,
Topical Application of 17β-Estradiol Increases Extracellular Matrix Protein Synthesis by Stimulating TGF-β Signaling in Aged Human Skin In Vivo  Eui Dong.

Topical Application of 17β-Estradiol Increases Extracellular Matrix Protein Synthesis by Stimulating TGF-β Signaling in Aged Human Skin In Vivo  Eui Dong.
Insulin-Like Growth Factor-I Enhances Transforming Growth Factor-β-Induced Extracellular Matrix Protein Production Through the P38/Activating Transcription.

Insulin-Like Growth Factor-I Enhances Transforming Growth Factor-β-Induced Extracellular Matrix Protein Production Through the P38/Activating Transcription.
IL-13-Stimulated Human Keratinocytes Preferentially Attract CD4+CCR4+ T cells: Possible Role in Atopic Dermatitis  Rahul Purwar, Thomas Werfel, Miriam.

IL-13-Stimulated Human Keratinocytes Preferentially Attract CD4+CCR4+ T cells: Possible Role in Atopic Dermatitis  Rahul Purwar, Thomas Werfel, Miriam.
Volume 60, Issue 5, Pages (November 2001)

Volume 60, Issue 5, Pages (November 2001)
Interleukin-17 and Interferon-γ Synergize in the Enhancement of Proinflammatory Cytokine Production by Human Keratinocytes  Marcel B.M. Teunissen, Jan.

Interleukin-17 and Interferon-γ Synergize in the Enhancement of Proinflammatory Cytokine Production by Human Keratinocytes  Marcel B.M. Teunissen, Jan.
IFN-γ Upregulates Expression of the Mouse Complement C1rA Gene in Keratinocytes via IFN-Regulatory Factor-1  Sung June Byun, Ik-Soo Jeon, Hyangkyu Lee,

IFN-γ Upregulates Expression of the Mouse Complement C1rA Gene in Keratinocytes via IFN-Regulatory Factor-1  Sung June Byun, Ik-Soo Jeon, Hyangkyu Lee,
Differential Expression of a Novel Gene in Response to hsp27 and Cell Differentiation in Human Keratinocytes  Mojgan Hell-Pourmojib, Peter Neuner, Robert.

Differential Expression of a Novel Gene in Response to hsp27 and Cell Differentiation in Human Keratinocytes  Mojgan Hell-Pourmojib, Peter Neuner, Robert.
A Fibrogenic Cytokine, Platelet-Derived Growth Factor (PDGF), Enhances Mast Cell Growth Indirectly Via a SCF- and Fibroblast-Dependent Pathway  Takaaki.

A Fibrogenic Cytokine, Platelet-Derived Growth Factor (PDGF), Enhances Mast Cell Growth Indirectly Via a SCF- and Fibroblast-Dependent Pathway  Takaaki.
Fas Ligand Downregulation with Antisense Oligonucleotides in Cells and in Cultured Tissues of Normal Skin Epidermis and Basal Cell Carcinoma  Jingmin.

Fas Ligand Downregulation with Antisense Oligonucleotides in Cells and in Cultured Tissues of Normal Skin Epidermis and Basal Cell Carcinoma  Jingmin.

Similar presentations

Ads by Google