ABSTRACT
Keratinocytes are frequently used to examine efficacy of wound healing products and dermatological agents in vitro. Cultured keratinocyte sheets are also used as autologous or allogenic grafts to promote wound closure. Because it is well known that the expression patterns of keratin genes change when cell cultures reach confluence, we investigated the expression pattern of wound healing-related genes, including growth factors and cytokines. Of additional particular interest is a novel wound healing related factor, secretory leukocyte protease inhibitor (SLPI), which appears to enhance tissue repair. We found that the expression pattern varied for specific genes expressed by keratinocytes as confluence was reached. Specifically, SLPI expression peaked in the early postconfluent state and vascular endothelial growth factor and amphiregulin in the late postconfluent state. Some gene products exhibit autocrine activity, whereas others exert paracrine regulation of growth. These findings indicate that it is critical to define the growth and differentiation state of human keratinocyte cultures to better determine responses and efficacy in vitro to various dermatological/wound care agents tested.
Subject(s)
Gene Expression/physiology , Keratinocytes/metabolism , Keratins/genetics , Wound Healing/physiology , Amphiregulin , Blotting, Northern , Cells, Cultured , EGF Family of Proteins , Glycoproteins/genetics , Humans , In Vitro Techniques , Intercellular Signaling Peptides and Proteins/genetics , Proteinase Inhibitory Proteins, Secretory , Proteins/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Secretory Leukocyte Peptidase Inhibitor , Transforming Growth Factor alpha/genetics , Vascular Endothelial Growth Factor A/geneticsABSTRACT
Chronic wounds are a major health care burden. Multiple factors produced by healing wounds play important roles in efficient and orderly wound healing. Secretory leukocyte protease inhibitor (SLPI) is constitutively expressed in epithelial cells, and its expression is increased by inflammation. SLPI has antimicrobial activities and improves wound healing. We hypothesized that SLPI expression correlates with keratinocyte growth rate and is increased by epidermal growth factor (EGF). Keratinocytes were isolated from neonatal foreskin. Subconfluent and confluent culture conditions were used. SLPI-specific primers were designed for use in quantitative-competitive reverse-transcription polymerase chain reaction assays to detect and quantify SLPI mRNA expression levels. SLPI levels were increased 2-fold in subconfluent cultures and 1.6-fold in confluent cultures to which EGF had been added. Confluent cultures also demonstrated 7-fold (growth factor depleted) and 6-fold (EGF) increase in SLPI RNA copy number, compared to subconfluent cultures. The results indicate that EGF increases SLPI expression.
