Osteoarthritis (OA) is a debilitating joint disease characterized by progressive cartilage degeneration, with no available disease-modifying therapy. OA is driven by pathological chondrocyte hypertrophy (CH), the cellular regulators of which are unknown. We have recently reported the therapeutic efficacy of G protein–coupled receptor kinase 2 (GRK2) inhibition in other diseases by recovering protective G protein–coupled receptor (GPCR) signaling. However, the role of GPCR-GRK2 pathway in OA is unknown. Thus, in a surgical OA mouse model, we performed genetic GRK2 deletion in chondrocytes or pharmacological inhibition with the repurposed U.S. Food and Drug Administration (FDA)–approved antidepressant paroxetine. Both GRK2 deletion and inhibition prevented CH, abated OA progression, and promoted cartilage regeneration. Supporting experiments with cultured human OA cartilage confirmed the ability of paroxetine to mitigate CH and cartilage degradation. Our findings present elevated GRK2 signaling in chondrocytes as a driver of CH in OA and identify paroxetine as a disease-modifying drug for OA treatment.
Extracting high-quality RNA from articular cartilage is challenging due to low cellularity and high proteoglycan content. This problem hinders efficient application of RNA sequencing (RNA-seq) analysis in studying cartilage homeostasis. Here we developed a method that purifies high-quality RNA directly from cartilage. Our method optimized the collection and homogenization steps so as to minimize RNA degradation, and modified the conventional TRIzol protocol to enhance RNA purity. Cartilage RNA purified using our method has appropriate quality for RNA-seq experiments including an RNA integrity number of ∼ 8. Our method also proved efficient in extracting high-quality RNA from subchondral bone.
Regenerating fish fins return to their original size and shape regardless of the nature or extent of injury. Prevailing models for this longstanding mystery of appendage regeneration speculate fin cells maintain uncharacterized positional identities that instruct outgrowth after injury. Using 45 zebrafish, we find differential Wnt production correlates with the extent of regeneration across the caudal fin. We identify Dachshund transcription factors as markers of distal blastema cells that produce Wnt and thereby promote a pro-progenitor and -proliferation environment. We show these Dach-expressing "niche cells" derive from mesenchyme populating cylindrical and progressively tapered fin rays. The niche pool, and consequently Wnt, steadily dissipates as 50 regeneration proceeds; once exhausted, ray and fin growth stops. Supported by mathematical modeling, we show longfin t2 zebrafish regenerate exceptionally long fins due to a perdurant niche, representing a "broken countdown timer". We propose regenerated fin size is dictated by the amount of niche formed upon damage, which simply depends on the availability of intra-ray mesenchyme defined by skeletal girth at the injury site. Likewise, the fin reestablishes a tapered 55 ray skeleton because progenitor osteoblast output reflects diminishing niche size. This "transpositional scaling" model contends mesenchyme-niche state transitions and positional information provided by self-restoring skeletal geometry rather than cell memories determine a regenerated fin's size and shape. 60 2 MAIN TEXTRegenerating organs restore their original size and shape after injury. Vertebrate appendage regeneration, including that of teleost fish fins, provides a striking example of this phenomenon. Major fin amputations, tiny resections, and cuts of diverse geometry all produce the same outcome -a restored fin matching the original's form and in scale with the animal's 65 body. Spallanzani, Broussonet, and T. H. Morgan pioneered studies of this longstanding mystery of regeneration in the 18 th and 19 th centuries (Broussonet, 1786;Morgan, 1900). For example, Morgan used oblique caudal fin resections to show that regeneration rates initially correlate with
40Organs stop growing to achieve the size and shape characteristic of the species and in scale with the animal's body. Likewise, regenerating organs sense injury extents to instruct appropriate replacement growth. Fish fins exemplify both phenomena through their tremendous diversity of form and remarkably robust regeneration. The classic zebrafish mutant longfin develops and regenerates dramatically elongated fins and underlying bony ray skeleton. We recently showed 45 longfin disrupts the orderly depletion of a growth-promoting blastema "niche" sub-population during fin regeneration. Initial niche sizes correlate with the amount of niche-generating intra-ray mesenchyme released from variably sized and tapered rays upon injury. Therefore, skeletal geometry-defined positional information and niche depletion dynamics can explain robust fin size restoration. Here, we find the longfin eponymous phenotype is entirely caused by cis over-50 expression of kcnh2a, a voltage-gated potassium channel related to human ether-a-go-go.Temporal delivery of a small molecule inhibitor confirms Kcnh2a actively extends the fin outgrowth period. We use blastula transplantations to show longfin-expressed kcnh2a acts tissue autonomously in the intra-ray mesenchyme/niche lineage, where it is concordantly ectopically expressed. We propose membrane potential dynamics and downstream ion signaling promote 55 niche-to-mesenchyme transitions to progressively slow outgrowth and thereby establish and restore fin size and shape.
Organs stop growing to achieve a characteristic size and shape in scale with the body of an animal. Likewise, regenerating organs sense injury extents to instruct appropriate replacement growth. Fish fins exemplify both phenomena through their tremendous diversity of form and remarkably robust regeneration. The classic zebrafish mutant longfint2 develops and regenerates dramatically elongated fins and underlying ray skeleton. We show longfint2 chromosome 2 overexpresses the ether-a-go-go-related voltage-gated potassium channel kcnh2a. Genetic disruption of kcnh2a in cis rescues longfint2, indicating longfint2 is a regulatory kcnh2a allele. We find longfint2 fin overgrowth originates from prolonged outgrowth periods by showing Kcnh2a chemical inhibition during late stage regeneration fully suppresses overgrowth. Cell transplantations demonstrate longfint2-ectopic kcnh2a acts tissue autonomously within the fin intra-ray mesenchymal lineage. Temporal inhibition of the Ca2+-dependent phosphatase calcineurin indicates it likewise entirely acts late in regeneration to attenuate fin outgrowth. Epistasis experiments suggest longfint2-expressed Kcnh2a inhibits calcineurin output to supersede growth cessation signals. We conclude ion signaling within the growth-determining mesenchyme lineage controls fin size by tuning outgrowth periods rather than altering positional information or cell-level growth potency.
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