Burns 29 (2003) 527–531 Matrix metalloproteinase expression in cytokine stimulated human dermal fibroblasts Mohan R.K. Dasu ∗ , Robert E. Barrow, Marcus Spies, David N. Herndon Department of Surgery, Shriners Hospitals for Children, University of Texas Medical Branch, 815 Market Street, Galveston, TX 77550, USA Accepted 29 April 2003 Abstract In this study, we investigated the effect of inflammatory cytokines on matrix metalloproteinase (MMP-1) and TIMP-1 production in human dermal fibroblasts, which play a pivotal role in wound healing, ranging from the synthesis and remodeling of extracellular matrix (ECM) to the synthesis of growth factors. The balance of MMPs and TIMPs is crucial in directing successful wound repair. Human adult dermal fibroblasts were seeded in six well plates (7.5 ×10 4 cells/ml) in complete media. Eighty to ninety percent confluent cells were treated with interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor- (TNF-) (10 ng/ml) for 6 h in serum free media with suitable controls run in triplicate. Supernatants were assayed for pro-MMP-1 & TIMP-1. Extracted total RNA was used for reverse transcription polymerase chain reaction (RT-PCR) with sequence specific primers for MMP-1, TIMP-1 and -actin. Signal intensity was normalized to the internal control (-actin). Statistical analysis used ANOVA. MMP-1 and TIMP-1 mRNA expression were markedly increased with IL-6 and TNF- treatment and remains unchanged with IL-1. Pro-MMP-1 protein levels are unchanged with TNF- and significantly increased with IL-1 and IL-6 treatment. However, TNF- significantly increases TIMP-1 protein levels. Data suggests differential regulation of MMP-1 and TIMP-1 protein levels by the cytokines found in stimulated dermal fibroblasts. Further characterization of this response will provide an understanding of the mechanisms of pathogenesis of extracellular matrix (ECM) and the potential role of metalloproteinases in tissue remodeling after injury. © 2003 Elsevier Science Ltd and ISBI. All rights reserved. Keywords: MMP-1; TIMP-1; Cytokines; Dermal fibroblasts; Extracellular matrix 1. Introduction Abnormal extracellular matrix deposition and delayed healing are characteristics of burn wounds. Matrix metal- loproteinases (MMPs) and their tissue inhibitors (TIMPs) play a major role in tissue regeneration and remodeling. MMPs are crucial in the regulation of cellular migration and extracellular matrix remodeling after injury. Matrix met- alloproteinases are zinc dependent endogenous proteases which are primarily secreted in the zymogen-inactive form [1]. Depending on their different substrates, matrix metal- loproteinases are divided into subgroups. After activation, MMPs cleave one or more components of the extracellular matrix [2,3]. MMP-1, is primarily produced by dermal fi- broblasts and has a high specificity for collagen types I and III. MMP-1 is secreted as an inactive 52 kDa pro-enzyme and proteolytically activated to the 42 kDa form. MMP-1 activity is stoichiometrically inhibited by TIMP-1, which is a specific tissue inhibitor [4]. ∗ Corresponding author. Tel.: +1-409-770-6703; fax: +1-409-770-6919. E-mail address: drmohan@utmb.edu (M.R.K. Dasu). TIMPs inhibit the proteolytic activity of MMPs [5]. The four different types of tissue inhibitors of metalloproteinases all form physiologically irreversible complexes with MMPs. A balance of MMPs and their tissue inhibitors are crucial in directing successful wound repair [6]. MMP regulation takes place at the transcriptional level through positive and negative regulatory elements and at the post-transcriptional level by proteolytic activation of MMP pro-enzymes and inactivation through TIMPs. Secretion and expression of MMP activation enzymes and TIMPs are influenced by cy- tokines [7]. Human dermal fibroblasts play a pivotal role in wound healing by synthesizing growth factors and remod- eling extracellular matrix. Fibroblast MMP production and activation are regulated by cytokines, growth factors and TIMPs. We hypothesize that derangements in the production of MMPs and/or its tissue inhibitors after injury are due to changes in inflammatory cytokines. To test this hypothesis, we investigated the effect of cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor- (TNF-) on MMP-1 and TIMP-1 production by human der- mal fibroblasts. 0305-4179/$30.00 © 2003 Elsevier Science Ltd and ISBI. All rights reserved. doi:10.1016/S0305-4179(03)00154-2