Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; Rosa26fs-tdTomato and Col10a1Cre; Rosa26fs-tdTomato) in combination with a PDGFRaH2B-GFP transgenic line, single cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage.

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Osteoarthritis (OA) and posttraumatic OA (PTOA) are caused by an imbalance in catabolic and anabolic processes in articular cartilage and proinflammatory changes throughout the joint, leading to joint degeneration and pain. We examined whether interleukin-6 (IL-6) signaling contributed to cartilage degradation and pain in PTOA. Genetic ablation of Il6 in male mice decreased PTOA-associated cartilage catabolism, innervation of the knee joint, and nociceptive signaling without improving PTOA-associated subchondral bone sclerosis or chondrocyte apoptosis. These effects were not observed in female Il6 −/− mice. Compared with wild-type mice, the activation of the IL-6 downstream mediators STAT3 and ERK was reduced in the knees and dorsal root ganglia (DRG) of male Il6 −/− mice after knee injury. Janus kinases (JAKs) were critical for STAT and ERK signaling in cartilage catabolism and DRG pain signaling in tissue explants. Whereas STAT3 signaling was important for cartilage catabolism, ERK signaling mediated neurite outgrowth and the activation of nociceptive neurons. These data demonstrate that IL-6 mediates both cartilage degradation and pain associated with PTOA in a sex-specific manner and identify tissue-specific contributions of downstream effectors of IL-6 signaling, which are potential therapeutic targets for disease-modifying OA drugs.

Focal adhesions (FAs) are highly dynamic structures that are assembled and disassembled on a continuous basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion and spreading to directed cell migration. The turnover of FAs is regulated at multiple levels and involves a variety of signaling molecules and adaptor proteins. In the present study, we show that in response to integrin engagement, a subcellular pool of Protein Kinase D1 (PKD1) localizes to the FAs. PKD1 affects FAs by decreasing turnover and promoting maturation, resulting in enhanced cell adhesion. The effects of PKD1 are mediated through direct phosphorylation of FA-localized phosphatidylinositol-4-phosphate 5-kinase type-l γ (PIP5Klγ) at serine residue 448. This phosphorylation occurs in response to Fibronectin-RhoA signaling and leads to a decrease in PIP5Klγs’ lipid kinase activity and binding affinity for Talin. Our data reveal a novel function for PKD1 as a regulator of FA dynamics and by identifying PIP5Klγ as a novel PKD1 substrate provide mechanistic insight into this process.

Although sea urchin gastrulation is well described at the cellular level, our understanding of the molecular changes that trigger the coordinated cell movements involved is not complete. Jun N-terminal kinase (JNK) is a component of the planar cell polarity pathway and is required for cell movements during embryonic development in several animal species. To study the role of JNK in sea urchin gastrulation, embryos were treated with JNK inhibitor SP600125 just prior to gastrulation. The inhibitor had a limited and specific effect, blocking invagination of the archenteron. Embryos treated with 2 μM SP600125 formed normal vegetal plates, but did not undergo invagination to form an archenteron. Other types of cell movements, specifically ingression of the skeletogenic mesenchyme, were not affected, although the development and pattern of the skeleton was abnormal in treated embryos. Pigment cells, derived from nonskeletogenic mesenchyme, were also present in SP600125-treated embryos. Despite the lack of a visible archenteron in treated embryos, cells at the original vegetal plate expressed several molecular markers for endoderm differentiation. These results demonstrate that JNK activity is required for invagination of the archenteron but not its differentiation, indicating that in this case, morphogenesis and differentiation are under separate regulation.