Induction of hyaluronan metabolism after mechanical injury of human peritoneal mesothelial cells in vitro

Abstract

Induction of hyaluronan metabolism after mechanical injury of human peritoneal mesothelial cells in vitro.BackgroundHyaluronan (HA) is an important extracellular matrix component that is involved in cell movement and tissue repair. In vertebrates, HA synthase genes (HAS 1, HAS 2, and HAS 3) that control the synthesis of HA have been identified. In this article, we investigated HA synthesis in the response of human peritoneal mesothelial cells (HPMCs) to injury.MethodsThe expression of HAS 1, HAS 2, and HAS 3 mRNA and the synthesis of [3H]-labeled HA were examined in an in vitro model of peritoneal mesothelial cell damage. The staining for uridine diphosphoglucose dehydrogenase, a key enzyme in the synthesis of HA, and biotinylated HA-binding protein was used to determine the cellular location of HA synthesis and its site of deposition.ResultsGrowth-arrested human HPMCs expressed low levels of mRNA for HAS 2 and HAS 3 but not HAS 1. Following injury to the monolayer, HAS 2 was up-regulated by 6 hours, reaching maximal expression between 12 and 24 hours. In contrast, the expression of HAS 3 was down-regulated. During the same time period, synthesis of HA was increased in the injured monolayer. This synthetic activity appeared to be restricted to cells at the edge of the wound and to cells entering the wound. In a separate series of experiments, the addition of HA to the injured monolayer at a concentration range found in peritoneal fluid (50 to 3300 ng/mL) increased the migration of cells into the wound in a dose-dependent manner.ConclusionsThese studies provide evidence that HA is an important component of peritoneal mesothelial cell migration. The results also suggest that in this process, there is differential regulation of HAS gene expression and that the synthesis of HA is limited to cells located at the leading edge of the wound. Induction of hyaluronan metabolism after mechanical injury of human peritoneal mesothelial cells in vitro. Hyaluronan (HA) is an important extracellular matrix component that is involved in cell movement and tissue repair. In vertebrates, HA synthase genes (HAS 1, HAS 2, and HAS 3) that control the synthesis of HA have been identified. In this article, we investigated HA synthesis in the response of human peritoneal mesothelial cells (HPMCs) to injury. The expression of HAS 1, HAS 2, and HAS 3 mRNA and the synthesis of [3H]-labeled HA were examined in an in vitro model of peritoneal mesothelial cell damage. The staining for uridine diphosphoglucose dehydrogenase, a key enzyme in the synthesis of HA, and biotinylated HA-binding protein was used to determine the cellular location of HA synthesis and its site of deposition. Growth-arrested human HPMCs expressed low levels of mRNA for HAS 2 and HAS 3 but not HAS 1. Following injury to the monolayer, HAS 2 was up-regulated by 6 hours, reaching maximal expression between 12 and 24 hours. In contrast, the expression of HAS 3 was down-regulated. During the same time period, synthesis of HA was increased in the injured monolayer. This synthetic activity appeared to be restricted to cells at the edge of the wound and to cells entering the wound. In a separate series of experiments, the addition of HA to the injured monolayer at a concentration range found in peritoneal fluid (50 to 3300 ng/mL) increased the migration of cells into the wound in a dose-dependent manner. These studies provide evidence that HA is an important component of peritoneal mesothelial cell migration. The results also suggest that in this process, there is differential regulation of HAS gene expression and that the synthesis of HA is limited to cells located at the leading edge of the wound.