The crucial trace element zinc stimulates osteogenesis in vitro and in vivo. However, the pathways mediating these effects remain poorly understood. This study aimed to investigate the effects of zinc on osteoblast differentiation in human bone marrow-derived mesenchymal stem cells (hBMSCs) and to identify the molecular mechanisms of these effects. In hBMSCs, zinc exposure resulted in a dose-dependent increase in osteogenesis and increased mRNA and protein levels of the master transcriptional factor RUNX2. Analyzing the upstream signaling pathways of RUNX2, we found that protein kinase A (PKA) signaling inhibition blocked zinc-induced osteogenic effects. Zinc exposure increased transcriptional activity and protein levels of phospho-CREB and enhanced translocation of phospho-CREB into the nucleus. These effects were reversed by H-89, a potent inhibitor of PKA. Moreover, zinc exposure led to dose-dependent increases in levels of intracellular cAMP. These findings indicate that zinc activates the PKA signaling pathway by triggering an increase in intracellular cAMP, leading to enhanced osteogenic differentiation in hBMSCs. Our results suggest that zinc exerts osteogenic effects in hBMSCs by activation of RUNX2 via the cAMP-PKA-CREB signaling pathway. Zinc supplementation may offer a promise as a potential pharmaceutical therapy for osteoporosis and other bone loss conditions.

Mitogen-activated protein kinases (MAPKs) are a family of protein kinases that function as key signal transducers of a wide spectrum of extracellular stimuli, including growth factors and pro-inflammatory cytokines. Dysregulation of the extracellular signal-regulated kinase (ERK) MAPK pathway is associated with human skeletal abnormalities including Noonan syndrome, neurofibromatosis type 1, and cardiofaciocutaneous syndrome. Here, we demonstrate that ERK activation in osteoprogenitors is required for bone formation during skeletal development and homeostasis. Deletion of Mek1 and Mek2, kinases upstream of ERK MAPK, in osteoprogenitors (Mek1^OsxMek2^−/−), resulted in severe osteopenia and cleidocranial dysplasia (CCD), similar to that seen in humans and mice with impaired RUNX2 function. Additionally, tamoxifen-induced deletion of Mek1 and Mek2 in osteoprogenitors in adult mice (Mek1^Osx-ERTMek2^−/−) significantly reduced bone mass. Mechanistically, this corresponded to decreased activation of osteoblast master regulators, including RUNX2, ATF4, and β-catenin. Finally, we identified potential regulators of osteoblast differentiation in the ERK MAPK pathway using unbiased phospho-mass spectrometry. These observations demonstrate essential roles of ERK activation in osteogenesis and bone formation.

The modified Broström procedure for chronic lateral ankle instability (CLAI) has presented outstanding clinical results. However, after the procedure, some patients with generalized ligamentous laxity have experienced a recurrence of ankle instability. To understand the effect of generalized ligamentous laxity on prognosis and risk of recurrence in a cohort of patients with CLAI after the modified Broström procedure. Cohort study; Level of evidence, 3. A total of 199 ankles from 188 patients underwent the modified Broström procedure for CLAI with a mean follow-up of 60.1 months (range, 48-108 months). Generalized ligamentous laxity was assessed in all patients. The Karlsson-Peterson ankle score (Karlsson score), talar tilt angle, and anterior displacement of the talus were used to evaluate clinical and radiological outcomes. Risk factors associated with clinical outcomes were evaluated using bivariate analysis and logistic regression analysis. Survival outcomes were compared using Kaplan-Meier analysis. Generalized ligamentous laxity was evident in 42 cases (21.1%). The average Karlsson score improved from 54.6 ± 7.1 preoperatively to 87.9 ± 7.2 at last follow-up (P < .001). The presence of generalized ligamentous laxity was significantly associated with poor clinical and radiological outcomes. The rates of clinical failure were 10.8% and 45.2% in the nonlaxity group and the laxity group, respectively (P < .001). According to bivariate analysis, generalized ligamentous laxity, syndesmosis widening, osteochondral lesion of the talus, high preoperative talar tilt angle (>15°), and high preoperative anterior displacement of the talus (>10 mm) were significantly associated with clinical failure. Multivariate logistic regression analysis revealed that generalized ligamentous laxity was the most important independent predictor of clinical failure after the modified Broström procedure. The cumulative success rates for the nonlaxity group were significantly superior to those for the laxity group in Kaplan-Meier curves (P < .001). Generalized ligamentous laxity is an independent predictor of poor outcomes and a risk factor of recurrent instability following the modified Broström procedure for CLAI.

Background: Zinc, an essential trace element, inhibits osteoclast differentiation in vitro and in vivo. The molecular mechanism for the inhibitory effect of zinc, however, is poorly understood. The purpose of this study was to investigate the effect of zinc and determine its molecular mechanism on receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in mouse bone marrow-derived monocyte cells (BMMs) and RAW264.7 cells.Results: In BMMs, zinc treatment during osteoclast differentiation decreased RANKL-induced osteoclast formation in a dose-dependent manner. We show that zinc suppressed the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Zinc also accumulated phospho-Nfatc1 (p-Nfatc1) in the cytosol in a dose-dependent manner and inhibited the translocation of Nfatc1 to the nucleus in RAW264.7 cells. Zinc suppressed the activities of Nfatc1 in the nucleus without changing the activities of NF-κB in RAW264.7 cells. In contrast, calcineurin activity decreased in response to zinc but its protein level was unchanged. RANKL-induced Ca²⁺oscillations were inhibited by zinc treatment, but phospho-phospholipase Cγ1 (p-PLCγ1), the upstream signaling molecule of Ca²⁺oscillations, was unaffected. Moreover, a constitutively active form of Nfatc1 obviously rescued suppression of osteoclastogenesis by zinc.Conclusions: Taken together, these results demonstrate for the first time that the inhibitory effect of zinc during osteoclastogesis is caused by suppressing the Ca²⁺-Calcineurin-NFATc1 signaling pathway. Thus, zinc may be a useful therapeutic candidate for the prevention of bone loss caused by NFATc1 activation in osteoclasts.