Estrogen and testosterone supplementation improves tendon healing and functional recovery after rotator cuff repair.

Failure of healing after rotator cuff repair (RCR) is common. The purpose of the current study was to evaluate the effect of systemic estrogen or testosterone supplementation on tendon healing after RCR. Seventy-two adult male mice were utilized for all experiments. The supraspinatus tendon was transected and repaired with 6-0 Prolene suture on the left shoulder of 51 animals. Mice were segregated into three groups postoperative: (1) vehicle group (VG; n = 18), (2) estrogen group (EST; n = 17), and (3) testosterone group (TST; n = 16). An unrepaired control group (unrepaired, n = 21) did not have surgery. Utilizing these animals, histological analysis, activity testing, biomechanical testing and RNA sequencing (RNA-seq) was performed. At 8 weeks post-RCR, TST, and EST supplementation improved the overall histologic structure of the repaired enthesis site. No differences in ultimate failure loads or stiffness were detected between VG, EST, and TST groups after biomechanical testing. RCR caused a reduction in wheel activity compared to unrepaired controls and supplementation with TST restored wheel activity. RNA-seq analysis indicated that estrogen and testosterone regulated different pathways associated with enthesis healing, including a suppression of inflammatory signaling. Supplementation with sex hormones improved the structure of the repaired tendon enthesis and significantly regulated expression of diverse pathways regulating multiple biological processes. Testosterone administration following RCR restored wheel activity without having a detrimental impact on biomechanical strength. Future human studies of sex hormone supplementation after RCR are warranted as supplementation in an animal model may improve tendon enthesis healing.

RNA helicase DDX21 mediates nucleotide stress responses in neural crest and melanoma cells.

The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo1. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(alnz24) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress.