Histatin‐1 is a salivary antimicrobial peptide involved in the maintenance of enamel and oral mucosal homeostasis. Moreover, Histatin‐1 has been shown to promote re‐epithelialization in soft tissues, by stimulating cell adhesion and migration in oral and dermal keratinocytes, gingival and skin fibroblasts, endothelial cells and corneal epithelial cells. The broad‐spectrum activity of Histatin‐1 suggests that it behaves as a universal wound healing promoter, although this is far from being clear yet. Here, we report that Histatin‐1 is a novel osteogenic factor that promotes bone cell adhesion, migration, and differentiation. Specifically, Histatin‐1 promoted cell adhesion, spreading, and migration of SAOS‐2 cells and MC3T3‐E1 preosteoblasts in vitro, when placed on a fibronectin matrix. Besides, Histatin‐1 induced the expression of osteogenic genes, including osteocalcin, osteopontin, and Runx2, and increased both activity and protein levels of alkaline phosphatase. Furthermore, Histatin‐1 promoted mineralization in vitro, as it augmented the formation of calcium deposits in both SAOS‐2 and MC3T3‐E1 cells. Mechanistically, although Histatin‐1 failed to activate ERK1/2, FAK, and Akt, which are signaling proteins associated with osteogenic differentiation or cell migration, it triggered nuclear relocalization of β‐catenin. Strikingly, the effects of Histatin‐1 were recapitulated in cells that are nonosteogenically committed, since it promoted surface adhesion, migration, and the acquisition of osteogenic markers in primary mesenchymal cells derived from the apical papilla and dental pulp. Collectively, these observations indicate that Histatin‐1 is a novel osteogenic factor that promotes bone cell differentiation, surface adhesion and migration, as crucial events required for bone tissue regeneration.

Muscle and bone are tightly integrated through mechanical and biochemical signals. Osteoclasts are cells mostly related to pathological bone loss; however, they also start physiological bone remodeling. Therefore, osteoclast signals released during bone remodeling could improve both bone and skeletal muscle mass. Extracellular ATP is an autocrine/paracrine signaling molecule released by bone and muscle cells. Then, in the present work, it was hypothesized that ATP is a paracrine mediator released by osteoclasts and leads to skeletal muscle protein synthesis. RAW264.7-derived osteoclasts were co-cultured in Transwell® chambers with flexor digitorum brevis (FDB) muscle isolated from adult BalbC mice. The osteoclasts at the upper chamber were mechanically stimulated by controlled culture medium perturbation, resulting in a two-fold increase in protein synthesis in FDB muscle at the lower chamber. Osteoclasts released ATP to the extracellular medium in response to mechanical stimulation, proportional to the magnitude of the stimulus and partly dependent on the P2X7 receptor. On the other hand, exogenous ATP promoted Akt phosphorylation (S473) in isolated FDB muscle in a time- and concentration-dependent manner. ATP also induced phosphorylation of proteins downstream Akt: mTOR (S2448), p70S6K (T389) and 4E-BP1 (T37/46). Exogenous ATP increased the protein synthesis rate in FDB muscle 2.2-fold; this effect was blocked by Suramin (general P2X/P2Y antagonist), LY294002 (phosphatidylinositol 3 kinase inhibitor) and Rapamycin (mTOR inhibitor). These blockers, as well as apyrase (ATP metabolizing enzyme), also abolished the induction of FDB protein synthesis evoked by mechanical stimulation of osteoclasts in the co-culture model. Therefore, the present findings suggest that mechanically stimulated osteoclasts release ATP, leading to protein synthesis in isolated FDB muscle, by activating the P2-PI3K-Akt-mTOR pathway. These results open a new area for research and clinical interest in bone-to-muscle crosstalk in adaptive processes related to muscle use/disuse or in musculoskeletal pathologies.

ObjectiveExtracellular nucleotides are released from different cells surrounding skeletal muscle, like motoneuron, endothelial cells, fibroblasts and bone cells during exercise. Even the muscle fibers themselves release ATP during membrane depolarization and contraction. Could this ATP‐enriched environment modulate protein synthesys in skeletal muscle? In this study, we assessed if extracellular ATP leads to protein synthesis in adult muscle through activation of Akt‐mTOR signaling pathway.MethodsFlexor Digitorium Brevis (FDB) muscles from 6–7 weeks‐old BalbC mice were dissected. Isolated muscles were stimulated with exogenous ATP in a time‐ and concentration‐dependent manner (3–20 min, 0.1 – 100 μM). The relative protein levels of p‐Akt(Ser473), p‐mTOR( Ser2448), p‐p70S6K(Thr389) and p‐4E‐BP1(Thr37/46) were evaluated by immunoblot. Normalization with the non‐phosphorylated protein and GAPDH was performed. The protein synthesis was estimated by the nonradioactive SUrface SEnsing of Translation (SUnSET) technique.Results100 μM ATP increased p‐Akt(Ser473) levels by 3‐fold in 10–15 min. Akt phosphorylation was concentration‐dependent, with a bell‐shaped curve that peaked in 3 μM ATP. Phosphorylation of Akt signalling‐related proteins (mTOR, p70S6K, and 4E‐BP1) and protein synthesis levels were also increased after 10–20 min with 3 μM ATP and UTP. Preincubation for 30 min with a general P2Y/P2X receptors antagonist (100 μM suramin), PI3‐kinase inhibitor (50 μM LY294002) or mTOR inhibitor (100 nM Rapamycin) abolished the effect of ATP on the protein synthesis.ConclusionThese findings suggest that extracellular ATP promotes protein synthesis in skeletal muscle through the activation of P2YRs and the Akt‐mTOR signaling pathway. Considering that effect on protein synthesis was mimicked by UTP (P2Y2R/P2Y4R agonist), and that P2Y2R is the mostly expressed nucleotide receptor in FDB muscle (as we previously described), a relevant role of P2Y2R in controlling protein synthesis in skeletal muscles is proposed. This proposed pathway could have a fine‐tuned role in the regulation of muscle mass during the integrated musculoskeletal activity.Support or Funding InformationFunded by Fondecyt‐1151353(SB)‐1151293(EJ)‐CONICYT‐PCHA‐63140009(CM)‐21151035(MA‐C).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fibroblast growth factor 21 (FGF21) is a hormone involved in the regulation of lipid, glucose, and energy metabolism. Although it is released mainly from the liver, in recent years it has been shown that it is a “myokine”, synthesized in skeletal muscles after exercise and stress conditions through an Akt-dependent pathway and secreted for mediating autocrine and endocrine roles. To date, the molecular mechanism for the pathophysiological regulation of FGF21 production in skeletal muscle is not totally understood. We have previously demonstrated that muscle membrane depolarization controls gene expression through extracellular ATP (eATP) signaling, by a mechanism defined as “Excitation-Transcription coupling”. eATP signaling regulates the expression and secretion of interleukin 6, a well-defined myokine, and activates the Akt/mTOR signaling pathway. This work aimed to study the effect of electrical stimulation in the regulation of both production and secretion of skeletal muscle FGF21, through eATP signaling and PI3K/Akt pathway. Our results show that electrical stimulation increases both mRNA and protein (intracellular and secreted) levels of FGF21, dependent on an extracellular ATP signaling mechanism in skeletal muscle. Using pharmacological inhibitors, we demonstrated that FGF21 production and secretion from muscle requires the activation of the P2YR/PI3K/Akt/mTOR signaling pathway. These results confirm skeletal muscle as a source of FGF21 in physiological conditions and unveil a new molecular mechanism for regulating FGF21 production in this tissue. Our results will allow to identify new molecular targets to understand the regulation of FGF21 both in physiological and pathological conditions, such as exercise, aging, insulin resistance, and Duchenne muscular dystrophy, all characterized by an alteration in both FGF21 levels and ATP signaling components. These data reinforce that eATP signaling is a relevant mechanism for myokine expression in skeletal muscle.