TSG-6 controls transcription and activation of matrix metalloproteinase 1 in conjunctivochalasis.
OBJECTIVE
To investigate the role of anti-inflammatory TSG-6 in controlling MMP-1 and MMP-3, which have been shown to be upregulated in conjunctivochalasis (CCh).


METHODS
Immunostaining of TSG-6 was compared between normal and CCh conjunctiva and Tenon's capsule. Second cultures of normal and CCh fibroblasts were transfected with or without TSG-6 siRNA and then with or without the addition of TNF-α or IL-1β. Cell lysates and culture media were collected to assess apoptosis with the use of ELISA and the expression of TSG-6, MMP-1, and MMP-3 transcripts and proteins with the use of qRT-PCR and Western blot analysis, respectively.


RESULTS
TSG-6 expression was constitutive in the in vivo normal conjunctival epithelium. Significantly more TSG-6-positive cells than normal specimens were noted in CCh subconjunctival tissue and Tenon's capsule. TSG-6 was constitutively expressed intracellularly by both resting normal and CCh fibroblasts but was secreted extracellularly only by resting CCh fibroblasts. Intracellular and extracellular TSG-6 proteins were markedly upregulated by TNF-α or IL-1β in normal and CCh fibroblasts. Active MMP-1 was found in CCh fibroblasts intracellularly and extracellularly, whereas only proMMP-1 was found intracellularly in normal fibroblasts. Knockdown by TSG-6 siRNA upregulated more MMP-1 than MMP-3 transcripts in normal and CCh fibroblasts. TSG-6 siRNA led to extracellular MMP-1 expression by normal fibroblasts such as CCh fibroblasts. This activation of MMP-1 was further enhanced by IL-1β. Cell apoptosis was higher in CCh fibroblasts and further aggravated by TSG-6 siRNA knockdown.


CONCLUSIONS
TSG-6 exerts an anti-inflammatory function by counteracting the transcription of MMP-1 and MMP-3 and the activation of MMP-1. Dysfunction of TSG-6 might play a role in the pathogenesis of CCh.
R&D Department, TissueTech, Inc., Ocular Surface Center, and Ocular Surface Research & Education Foundation, Miami, Florida, USA.
Ping
P
Guo
Su-Zhen
SZ
Zhang
Hua
H
He
Ying-Ting
YT
Zhu
Scheffer C G
SC
Tseng
D000893:Anti-Inflammatory Agents / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured; D003229:Conjunctival Diseases / Q000201:enzymology* / Q000378:metabolism; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000378:metabolism*; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction; D015533:Transcriptional Activation / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation
D000893:Anti-Inflammatory Agents; D015815:Cell Adhesion Molecules; D053583:Interleukin-1beta; C072273:TNFAIP6 protein, human; D014409:Tumor Necrosis Factor-alpha; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1