MicroRNAs (miRNAs) play key roles in cartilage development and homeostasis and are dysregulated in osteoarthritis. MiR-145 modulation induces profound changes in the human articular chondrocyte (HAC) phenotype, partially through direct repression of SOX9. Since miRNAs can simultaneously silence multiple targets, we aimed to identify the whole targetome of miR-145 in HACs, critical if miR-145 is to be considered a target for cartilage repair. We performed RIP-seq (RNA-immunoprecipitation and high-throughput sequencing) of miRISC (miRNA-induced silencing complex) in HACs overexpressing miR-145 to identify miR-145 direct targets and used cWords to assess enrichment of miR-145 seed matches in the identified targets. Further validations were performed by RT-qPCR, Western immunoblot, and luciferase assays. MiR-145 affects the expression of over 350 genes and directly targets more than 50 mRNAs through the 3′UTR or, more commonly, the coding region. MiR-145 targets DUSP6, involved in cartilage organization and development, at the translational level. DUSP6 depletion leads to MMP13 upregulation, suggesting a contribution towards the effect of miR-145 on MMP13 expression. In conclusion, miR-145 directly targets several genes involved in the expression of the extracellular matrix and inflammation in primary chondrocytes. Thus, we propose miR-145 as an important regulator of chondrocyte function and a new target for cartilage repair.

PTHrP (parathyroid hormone-related protein) is crucial for normal cartilage development and long bone growth and acts to delay chondrocyte hypertrophy and terminal differentiation in the growth plate. After growth plate closure adult HACs (human articular chondrocytes) still produce PTHrP, suggesting a possible role for this factor in the permanent articular cartilage. However, the expression regulation and function of PTHrP in the permanent articular cartilage is unknown. Human articular cartilage is an avascular tissue and functions in a hypoxic environment. The resident chondrocytes have adapted to hypoxia and use it to drive their tissue-specific functions. In the present study, we explored directly in normal articular chondrocytes isolated from a range of human donors the effect of hypoxia on PTHrP expression and whether PTHrP can regulate the expression of the permanent articular chondrocyte phenotype. We show that in HACs PTHrP is up-regulated by hypoxia in a HIF (hypoxia-inducible factor)-1α and HIF-2α-dependent manner. Using recombinant PTHrP, siRNA-mediated depletion of endogenous PTHrP and by blocking signalling through its receptor [PTHR1 (PTHrP receptor 1)], we show that hypoxia-induced PTHrP is a positive regulator of the key cartilage transcription factor SOX9 [SRY (sex determining region on the Y chromosome)-box 9], leading to increased COL2A1 (collagen type II, α1) expression. Our findings thus identify PTHrP as a potential factor for cartilage repair therapies through its ability to promote the differentiated HAC phenotype.

SignificanceHow species differ from each other is a key question in biology. However, genetic mapping between species often fails because of sterile hybrid crosses. Here, we have developed a technique called in vitro recombination to circumvent breeding. We induced genetic reshuffling through mitotic recombination with the drug ML216 and mapped trait variations in a dish. Starting with hybrid embryonic stem cells between the Mus musculus laboratory mouse and Mus spretus spanning ∼1.5 million years of divergence, we show that it is possible to map the gene responsible for differential resistance to the drug tioguanine in as few as 21 days. Our technique opens up experimental avenues in genetic mapping of various traits and diseases across mouse species.

Discovering the genetic changes underlying species differences is a central goal in evolutionary genetics. However, hybrid crosses between species in mammals often suffer from hybrid sterility, greatly complicating genetic dissection of trait variation. Here we describe a simple, robust and transgene-free technique to make “in vitro crosses” in hybrid mouse embryonic stem cells by inducing random mitotic crossovers with the drug ML216, which inhibits Bloom syndrome (BLM). Starting with an interspecific hybrid (between Mus musculus and Mus spretus) embryonic stem cell line spanning 1.5 million years of divergence, we demonstrate the feasibility of mapping enzymatic differences across species within weeks and the possibility of re-deriving whole mice. Our work shows how in vitro crosses can overcome major bottlenecks like hybrid sterility in traditional mouse breeding to address fundamental questions in evolutionary biology.Impact StatementBy mixing hybrid mouse genomes in stem cells via mitotic recombination, genetic mapping and hybrid mosaic mice can be achieved in weeks, even across species barriers.