Podocalyxin and ciliary neurotrophic factor receptor are novel components of the surfaceome of chondrogenic cells

Osteoarthritis is a degenerative joint disease characterized by progressive loss of articular cartilage and limited capacity for intrinsic repair. A major barrier to developing effective regenerative strategies is the incomplete understanding of the molecular mechanisms regulating chondrogenesis and cartilage maintenance. Cell surface proteins are key mediators of extracellular communication, adhesion, and signaling, yet the chondrogenic surfaceome remains incompletely mapped, with prior studies focusing primarily on mature or cytokine-activated chondrocytes. The aim of this study was to provide a temporal profile of the surfaceome during in vitro chondrogenic differentiation and to identify novel membrane proteins with potential roles in cartilage biology. We applied a sialoglycoprotein-targeted glycocapture strategy to selectively enrich plasma membrane proteins from chick embryonic limb bud-derived micromass cultures undergoing chondrogenesis. Enriched samples underwent high-resolution shotgun proteomic analysis, and differentially expressed candidates were validated by western blotting, immunocytochemistry, and transient gene silencing. Functional effects on extracellular matrix gene regulation were assessed by quantitative RT-PCR and matrix histochemistry. This approach generated the temporal surfaceome map of chondrogenic progenitors. Among identified candidates, two proteins not previously linked to chondrogenesis, podocalyxin (PODXL) and ciliary neurotrophic factor receptor (CNTFR), were detected at the plasma membrane and confirmed at the protein and transcript levels. Both proteins exhibited time-dependent downregulation during differentiation. Targeted knockdown revealed differential regulation of the fibrocartilage marker COL1A1 expression, indicating non-redundant roles in cell-matrix signaling and survival pathways. Single-cell transcriptomic meta-analysis confirmed expression of both proteins in discrete human articular chondrocyte subpopulations. This study expands the molecular framework of chondrogenesis, identifying PODXL and CNTFR as novel, temporally regulated surfaceome components with distinct roles in extracellular matrix signaling. These findings complement prior proteomic analyses of cytokine-activated mature articular chondrocytes and suggest new candidates for developmental cartilage biomarkers and therapeutic targets for osteoarthritis. Our results provide a resource for future cross-species surfaceome studies and highlight key pathways for further investigation into cartilage lineage specification and matrix adaptation. Osteoarthritis is the most common joint disease worldwide, causing pain, stiffness, and reduced mobility as the smooth cartilage that covers the ends of bones gradually breaks down. Cartilage has a very limited ability to heal itself, and there are currently no treatments that can stop or reverse the disease. Scientists are working on new ways to repair or replace damaged cartilage, but to do this effectively, they need to understand the molecular “language” that cartilage‑forming cells use to develop and maintain healthy tissue. In this study, we examined the set of proteins that are present on the surface of cartilage‑forming cells, known as the “surfaceome.” These surface proteins play important roles in how cells connect to their surroundings, receive signals, and work together to build the cartilage matrix. Using a method to selectively capture and analyze these proteins, we discovered two that had never been linked to cartilage development: podocalyxin and ciliary neurotrophic factor receptor. We found that these two proteins may have opposite effects on cartilage quality. Because they can be detected on the outside of cells, they could be developed into markers for early osteoarthritis detection or as targets for new therapies. Our findings open the door to new strategies for diagnosing and treating joint disease by focusing on cell‑surface communication.