The gut epithelium has remarkable self-renewal capacity that under homeostatic conditions is driven by Wnt signalling in Lgr5(+) intestinal stem cells (ISCs). However, the mechanisms underlying ISC regeneration after injury remain poorly understood. The Hippo signalling pathway mediates tissue growth and is important for regeneration. Here we demonstrate in mice that Yap, a downstream transcriptional effector of Hippo, is critical for recovery of intestinal epithelium after exposure to ionizing radiation. Yap transiently reprograms Lgr5(+) ISCs by suppressing Wnt signalling and excessive Paneth cell differentiation, while promoting cell survival and inducing a regenerative program that includes Egf pathway activation. Accordingly, growth of Yap-deficient organoids is rescued by the Egfr ligand epiregulin, and we find that non-cell-autonomous production of stromal epiregulin may compensate for Yap loss in vivo. Consistent with key roles for regenerative signalling in tumorigenesis, we further demonstrate that Yap inactivation abolishes adenomas in the Apc(Min) mouse model of colon cancer, and that Yap-driven expansion of Apc(-/-) organoids requires the Egfr module of the Yap regenerative program. Finally, we show that in vivo Yap is required for progression of early Apc mutant tumour-initiating cells, suppresses their differentiation into Paneth cells, and induces a regenerative program and Egfr signalling. Our studies reveal that upon tissue injury, Yap reprograms Lgr5(+) ISCs by inhibiting the Wnt homeostatic program, while inducing a regenerative program that includes activation of Egfr signalling. Moreover, our findings reveal a key role for the Yap regenerative pathway in driving cancer initiation.

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Intervertebral disc degeneration (IVDD) is one of the key predisposing factors for low back pain. Although the exact mechanism remains unclear, inflammatory response and nucleus pulposus (NP) apoptosis are known to play important roles in this process. Prolactin protects against inflammation-associated chondrocyte apoptosis in arthritis. Based on prior studies, we hypothesized that prolactin might have therapeutic effects on IVDD by inhibiting the apoptosis of degenerative human disc NP cells. An experimental model of IVDD was established in 3-month-old Sprague-Dawley rats by submitting them to percutaneous disc puncture with a 20-gauge needle on levels 7–8 and 8–9 of the coccygeal vertebrae. Then the rats were injected with 20 or 200 ng prolactin on a weekly basis. Radiologic and histologic analyses were performed on days 4, 7, 14, and 28. The expression of prolactin and its receptor was analyzed in human tissue obtained from symptomatic patients undergoing microencoscopy discectomy, or from scoliosis patients undergoing deformity correction surgery. The results showed that intradiscal injection of prolactin maintained disc height and the mean signal intensity of the punctured disc. Histological analysis indicated that prolactin treatment significantly retained the complete structure of the NP and annulus fibrosus compared with the vehicle group. In addition, more collagen II, but fewer collagen I-containing tissues were detected in the prolactin treatment groups compared to the vehicle group. Moreover, low levels of tumor necrosis factor-α, interleukin-1β, cleaved-caspase 3, and TUNEL staining were observed in the prolactin treatment groups. We also demonstrated that prolactin impaired puncture-induced inflammation and cell apoptosis by downregulating activation of the NF-κB pathway. The degenerated NP tissues from patients had decreased expression of prolactin and its receptor, whereas expression was increased in the NP tissues removed from scoliosis patients. These results suggest that prolactin may be a novel therapeutic target for the treatment of IVDD.

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Intervertebral disc (IVD) degeneration (IDD) is a major cause of low back pain. The pathogenesis of IDD is associated with the disturbance of reactive oxygen species (ROS) equilibrium, inflammation, and matrix loss. Aspirin is a nonsteroidal anti-inflammatory drug that effectively inhibits inflammation and oxidative stress and has been widely used for the treatment of back pain. Therefore, we hypothesize that aspirin reverses the IDD process via antioxidative and anti-inflammatory effects on the AMPK signaling pathway. In vitro, aspirin diminished cellular oxygen free radicals (ROS, nitric oxide (NO)) and inflammatory cytokines (interleukin- (IL-) 1β and IL-6 and tumor necrosis factor alpha (TNF-α)) induced by lipopolysaccharides (LPS) in nucleus pulposus cells (NPCs). We found that aspirin preserved the extracellular matrix (ECM) content of collagen type II (COL2) and aggrecan while inhibiting the expression of matrix-degenerating enzymes, including matrix metalloproteinase 3 and 13 (MMP-3 and MMP-13) and A disintegrin and metalloproteinase with thrombospondin motifs 4 and 5 (ADAMTS-4, ADAMTS-5). Aspirin significantly promoted the ratios of p-AMPK to AMPK and p-ACC to ACC expression in NPCs. Furthermore, pretreatment with the AMPK inhibitor compound C abrogated the antioxidant effects of aspirin. In vivo, an IDD model was established in Sprague-Dawley rats via percutaneous disc puncture with the 20-gauge needle on levels 8-9 and 9-10 of the coccygeal vertebrae. Imaging assessment showed that after aspirin treatment, improvements in disc height index (DHI) ranged from 1.22-fold to 1.54-fold and nucleus pulposus signal strength improved from 1.26-fold to 1.33-fold. Histological analysis showed that aspirin treatment prevented the loss of COL2 and decreased MMP-3 and MMP-13, inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), IL-1β, and TNF-α expression in the IVD tissues. These results suggest that treatment with aspirin could reverse the IDD process via the AMPK signaling pathway, which provides new insights into the potential clinical applications of aspirin, particularly for IDD treatment.

Bone mass loss around prostheses is a major cause of implant failure, especially in postmenopausal osteoporosis patients. In osteoporosis, excess oxidative stress largely contributed abnormal bone remodeling. Melatonin, which is synthesized from the pineal gland, promotes osteoblast differentiation and bone formation and has effectively been used to combat oxidative stress. Thus, we determined if melatonin can inhibit oxidative stress to promote osteogenesis and improve bone mass around prostheses in osteoporosis. In this study, we observed that received melatonin at 50 mg/kg body weight significantly increased periprosthetic bone mass as well as implant fixation intensity in ovariectomized (OVX) rats. Meanwhile, it decreased the expression of oxidative stress markers (NAPDH oxidase 2 and cytochrome c) and enhanced expressing level of the formation markers of bones (alkaline phosphatase, osteocalcin, and osterix) around prostheses compared to that in the control group. Additionally, melatonin decreased hydrogen peroxide- (H2O2-) induced oxidative stress and restored the osteogenesis potential of MC3T3-E1 cells. Mechanistically, melatonin clearly increased mitochondrial sirtuin 3 (SIRT3) expression and decreased the ratio of acetylated superoxide dismutase 2 (AC-SOD2)/SOD2 compared to the H2O2 group. SIRT3 inhibition counteracted the protective effects of melatonin on oxidative stress and bone formation. Together, the results showed that melatonin ameliorated oxidative stress in mitochondrial via the SIRT3/SOD2 signaling pathway, thereby promoting osteogenesis, improving bone mass around the prostheses, and increasing initial stability. Thus, melatonin might be a suitable candidate to decrease the rate of implant failure and lengthen the lifespan of prostheses after total joint arthroplasty.

Mesenchymal stem cells (MSCs) hold great potential to treat tissue damage based on their multipotent property, and are also considered as suitable cell resources to create tissue-engineered grafts for tendon repair. However, the clinical application of MSCs is still limited by the lack of efficient methods to induce tenogenic differentiation. In this study, by performing the experiments in transwell system, we found that paracrine factors from tenocytes could induce MSCs to undergo the tenogenic differentiation. We further verified that tenocytes could secrete exosomes and these tenocyte-derived exosomes efficiently initiated the tenogenic differentiation of MSCs. Finally, we revealed that the TGF-b existing in tenocyte-derived exosomes mediated the process, as the inhibition of TGF-b signaling abolished the effects of tenocyte-derived exosomes on MSCs. By investigating the effects of tenocytes on MSCs, we found that tenocytes-derived exosomes can induce tenogenic differentiation of MSCs in a TGF-b dependent manner. These studies provided critical information about the multipotency of MSCs and suggested potential strategies for clinical translation.

Wear debris-induced osteogenic inhibition and bone destruction are critical in the initiation of peri-prosthetic osteolysis. However, the molecular mechanism underlying this phenomenon is poorly understood. In this study, we analyzed the involvement of the GSK-3β/β-catenin signal pathway, which is important for bone formation in this pathological condition. We established a titanium (Ti) particle-stressed murine MC3T3-E1 cell culture system and calvariae osteolysis model to test the hypothesis that Ti particle-induced osteogenic inhibition and bone destruction are mediated by the GSK-3β/β-catenin signal pathway. Our findings showed that Ti particles reduced osteogenic differentiation induced by osteogenesis-related gene expression, alkaline phosphatase activity and matrix mineralization, as well as pSer9-GSK-3β expression and β-catenin signal activity. Downregulation of GSK-3β activity attenuated Ti particle-induced osteogenic inhibition, whereas the β-catenin inhibitor reversed this protective effect. Moreover, the GSK-3β/β-catenin signal pathway mediated the upregulation of RANKL and downregulation of OPG in Ti particle-stressed MC3T3-E1 cells. In addition, our in vivo results showed that Ti particles induced bone loss via regulating GSK-3β and β-catenin signals. Based on these results, we concluded that the GSK-3β/β-catenin signal pathway mediates the adverse effects of Ti particles on osteoblast differentiation and bone destruction, and can be used as a potential therapeutic target for the treatment of peri-prosthetic osteolysis.