Cytokine-like 1 (CYTL1) Regulates the Chondrogenesis of Mesenchymal Cells

Jae-Sung Kim,Zae Young Ryoo,Jang-Soo Chun

doi:10.1074/jbc.m700965200

Abstract

To identify novel molecules regulating chondrogenesis and cartilage development, we screened a cartilage-specific expressed sequence tag data base. Cytokine-like 1 (Cytl1), a possible cytokine candidate with unknown function that was originally identified in bone marrow-derived CD34-positive cells, was selected for functional characterization. In view of the initial observation that Cytl1 is predominantly expressed in chondrocytes and cartilage, we investigated its possible role in chondrogenesis and hypertrophic maturation of chondrocytes. Cytl1 expression was very low in mesenchymal cells, dramatically increased during chondrogenesis, and decreased during hypertrophic maturation, both in vivo and in vitro. The role of Cytl1 in chondrogenesis and hypertrophic maturation was examined by treating chondrifying mesenchymal cells with exogenous Cytl1 or ectopic expression of Cytl1. Notably, exogenous Cytl1 caused chondrogenic differentiation of mouse limb bud mesenchymal cells during micromass culture. Lentivirus-mediated overexpression of Cytl1 additionally induced chondrogenic differentiation of mesenchymal cells. However, Cytl1 did not affect the hypertrophic maturation of chondrocytes. Cytl1 exerted its chondrogenic effect via stimulation of Sox9 transcriptional activity. In addition, Cytl1 caused expression of insulin-like growth factor 1, which has a capacity to induce chondrogenesis. Thus, our results collectively suggest that chondrocyte-specific Cytl1 regulates chondrogenesis as a novel autocrine factor, but not hypertrophic maturation of chondrocytes during cartilage development.

Highlights

Dral ossification, which requires the maturation of hypertrophic chondrocytes [1,2,3,4]
Cytokine-like 1 (Cytl1) Induces Chondrogenesis of Mesenchymal Cells without Effects on Hypertrophic Maturation— Because the chondrocyte-specific expression of Cytl1 suggests a possible function in chondrogenesis and/or hypertrophic maturation, developing ulna, Cytl1 transcript was detected in the region we examined its role in chondrogenic differentiation of mesenwhere collagen IIB is expressed, but it was not detected in the chymal cells and hypertrophic maturation of chondrocytes
We demonstrate in this study that Cytl1 expression is strongly correlated with chondrocyte differentiation, both in structural characteristics are common in cytokines [26, 27], leading to the assumption that Cytl1 is a cytokine-like protein

Summary

Primer sequencea

Antisense 5Ј-tgt agg cca tga ggt cca c-3Ј a Primers are designed from known mouse sequences. b AT means annealing temperature. Expression Vectors and Reporter Gene Assay—Cytl cDNA (GenBankTM accession number EF108311) was subjected to RT-PCR amplification from mouse rib chondrocytes using specific primers (Table 1) designed to introduce EcoRI and XhoI restriction sequences at the 5Ј and 3Ј ends of the amplified fragment, respectively. The cDNA fragments of mouse collagen IIB and collagen X were amplified by PCR using specific primers designed to introduce HindIII (sense) and EcoRI (antisense) restriction sequences at the 5Ј end (Table 1). Digested collagen IIB and collagen X and Cytl cDNA sequences were cloned into the pSPT-18 vector (Roche Diagnostics), linearized with EcoRI or HindIII digestion, and transcribed with SP6 and T7 RNA polymerase to generate sense and antisense probes, respectively. Alignment of Cytl amino acid sequences were filtered with Bioworks version 3.1

RESULTS

Findings

DISCUSSION