Bone morphogenetic protein-mediating receptor-associated Smads as well as common Smad are expressed in human articular chondrocytes but not up-regulated or down-regulated in osteoarthritic cartilage

Abstract

Bone morphogenetic proteins (BMPs) are supposed to be important for cartilage matrix anabolism. In this study, we investigated whether the intracellular mediators of BMP activity, Smads 1, 4, 5, and 8, are expressed in normal human articular chondrocytes in vivo and in vitro and whether alterations in expression and distribution pattern are found in osteoarthritic cartilage or in vitro after stimulation with interleukin (IL)-1, because down-regulation of these mediators could be responsible for the decrease of anabolic activity in osteoarthritic cartilage. RNA was isolated from normal and osteoarthritic human knee cartilage and analyzed by (quantitative) polymerase chain reaction (PCR) technology. Articular chondrocytes were cultured in alginate beads and short-term high-density monolayer cultures with and without stimulation by IL-1. In addition, immunolocalization of the receptor-associated Smads (R-Smads) was performed on sections of normal and diseased articular cartilage. Reverse-transcription (RT)-PCR analysis showed a moderate expression of all Smads investigated in normal, early degenerative, and late stage osteoarthritic cartilage. Immunolocalization detected the R-Smads in most chondrocytes on the protein level in all specimen groups investigated. In vitro, the Smads were also expressed and partly up-regulated by Il-1beta in alginate bead culture. Of note, for Smad 1, two truncated splice variants were expressed by articular chondrocytes missing exon 4 as well as exons 3 and 4. Our study showed that BMP-receptor Smads 1, 5, and 8 as well as common Smad (C-Smad) 4 are expressed and present in human normal and osteoarthritic articular chondrocytes corroborating the importance of BMPs and BMP signaling for articular cartilage. This study is the first to describe splicing variants for Smad 1. Smads 1, 4, and 5 are up-regulated in vitro by Il-1beta, suggesting a linkage of the Il-1 and BMP-signaling pathways within the chondrocytes. None of the Smads were grossly up- or down-regulated in osteoarthritic chondrocytes, suggesting that differences in overall expression levels of the investigated Smad proteins are not relevant for metabolic activity of articular chondrocytes in vivo.