Klotho Overexpression Is Frequently Associated With Upstream Rearrangements in Fusion-Negative Phosphaturic Mesenchymal Tumors of Bone and Sinonasal Tract.

Phosphaturic mesenchymal tumors (PMT) are uncommon neoplasms that cause hypophosphatemia/osteomalacia mainly by secreting fibroblast growth factor 23. We previously identified FN1::FGFR1/FGF1 fusions in nearly half of the PMTs and frequent KL (Klotho or α-Klotho) overexpression in only those with no known fusion. Here, we studied a larger cohort of PMTs for KL expression and alterations. By FN1 break-apart fluorescence in situ hybridization (FISH) and reappraisal of previous RNA sequencing data, 6 tumors previously considered "fusion-negative" (defined by negative results of FISH for FN1::FGFR1 fusion and FGF1 break-apart and/or of RNA sequencing) were reclassified as fusion-positive PMTs, including 1 containing a novel FN1::ZACN fusion. The final cohort of fusion-negative PMTs included 33 tumors from 32 patients, which occurred in the bone (n = 18), soft tissue (n = 10), sinonasal tract (n = 4), and brain (n = 1). In combination with previous work, RNA sequencing, RNA in situ hybridization, and immunohistochemistry showed largely concordant results and demonstrated KL/α-Klotho overexpression in 17 of the 28 fusion-negative and none of the 10 fusion-positive PMTs studied. Prompted by a patient in this cohort harboring germline KL upstream translocation with systemic α-Klotho overexpression and multifocal PMTs, FISH was performed and revealed KL rearrangement in 16 of the 33 fusion-negative PMTs (one also with amplification), including 14 of the 17 cases with KL/α-Klotho overexpression and none of the 11 KL/α-Klotho-low fusion-negative and 11 fusion-positive cases studied. Whole genomic sequencing confirmed translocation and inversion in 2 FISH-positive cases involving the KL upstream region, warranting further investigation into the mechanism whereby these rearrangements may lead to KL upregulation. Methylated DNA immunoprecipitation and sequencing suggested no major role of promoter methylation in KL regulation in PMT. Interestingly, KL-high/-rearranged cases seemed to form a clinicopathologically homogeneous group, showing a predilection for skeletal/sinonasal locations and typically matrix-poor, cellular solitary fibrous tumor-like morphology. Importantly, FGFR1 signaling pathways were upregulated in fusion-negative PMTs regardless of the KL status compared with non-PMT mesenchymal tumors by gene set enrichment analysis, perhaps justifying FGFR1 inhibition in treating this subset of PMTs.

Mechanisms of Nifedipine-Downregulated CD40L/sCD40L Signaling in Collagen Stimulated Human Platelets.

The platelet-derived soluble CD40L (sCD40L) release plays a critical role in the development of atherosclerosis. Nifedipine, a dihydropyridine-based L-type calcium channel blocker (CCB), has been reported to have an anti-atherosclerotic effect beyond its blood pressure-lowering effect, but the molecular mechanisms remain unclear. The present study was designed to investigate whether nifedipine affects sCD40L release from collagen-stimulated human platelets and to determine the potential role of peroxisome proliferator-activated receptor-ß/-γ (PPAR-ß/-γ). We found that treatment with nifedipine significantly inhibited the platelet surface CD40L expression and sCD40L release in response to collagen, while the inhibition was markedly reversed by blocking PPAR-ß/-γ activity with specific antagonist such as GSK0660 and GW9662. Meanwhile, nifedipine also enhanced nitric oxide (NO) and cyclic GMP formation in a PPAR-ß/-γ-dependent manner. When the NO/cyclic GMP pathway was suppressed, nifedipine-mediated inhibition of sCD40L release was abolished significantly. Collagen-induced phosphorylation of p38MAPK, ERK1/2 and HSP27, matrix metalloproteinase-2 (MMP-2) expression/activity and reactive oxygen species (ROS) formation were significantly inhibited by nifedipine, whereas these alterations were all attenuated by co-treatment with PPAR-ß/-γ antagonists. Collectively, these results demonstrate that PPAR-ß/-γ-dependent pathways contribute to nifedipine-mediated downregulation of CD40L/sCD40L signaling in activated platelets through regulation of NO/ p38MAPK/ERK1/2/HSP27/MMP-2 signalings and provide a novel mechanism regarding the anti-atherosclerotic effect of nifedipine.

Mechanisms of antiplatelet activity of nifedipine: role of peroxisome proliferator-activated receptor-ß-γ-dependent processes.

OBJECTIVE: Nifedipine, an L-type calcium channel blocker, is widely used in the treatment of hypertension and coronary heart diseases, and also exhibits an antiplatelet activity. Activation of peroxisome proliferator-activated receptors (PPARs; α, ß/δ, and γ) inhibits the platelet aggregation. Therefore, the purpose of this study was to evaluate the contribution of PPAR-mediated processes to the antiplatelet activity of nifedipine. METHODS AND RESULTS: We assessed human platelet aggregation by using an aggregometer and measured several platelet activating markers and related signaling pathways in platelets treated with nifedipine in the presence or absence of PPAR agonists. Nifedipine treatment (1, 5  µmol/l) dose-dependently increased the activity and intracellular expression of PPAR-ß/-γ by inhibiting the release of PPAR-ß/-γ from activated platelets. Nifedipine treatment also upregulated cyclic 3',5'-cyclic monophosphate (GMP)/protein kinase G (PKG) expression, and increased PI(3)K/Akt pathway, endothelial nitric oxide synthase, and soluble guanylyl cyclase activities. In the presence of a selective PPAR-ß antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibitory effects of nifedipine on collagen-induced platelet aggregation, intracellular Ca mobilization, and protein kinase C (PKC-α) activation were abrogated. Similarly, PPAR-ß-γ antagonists markedly attenuated nifedipine-mediated upregulation of nitric oxide/cyclic GMP/PKG cascade. In a mouse model of thrombosis, the administration of nifedipine substantially inhibited fluorescein sodium-induced vessel thrombus formation; however, the antithrombotic effect was considerably reduced in the presence of PPAR-ß/-γ antagonists. CONCLUSION: This study is the first to show that the PPAR-ß/-γ-dependent upregulation of PI(3)K/Akt/nitric oxide/cyclic GMP/PKG pathway and the inhibition of PKC-α activity and intracellular Ca(+) mobilization in platelets may be the mechanisms underlying the antiplatelet and antithrombotic activities of nifedipine.

The antiplatelet activity of magnolol is mediated by PPAR-ß/γ.

Activation of peroxisome proliferator-activated receptor (PPAR) isoforms (α, ß/δ, and γ) is known to inhibit platelet aggregation. In the present study, we examined whether PPARs-mediated pathways contribute to the antiplatelet activity of magnolol, a compound purified from Magnolia officinalis. Magnolol (20-60 µM) dose-dependently enhanced the activity and intracellular level of PPAR-ß/γ in platelets. In the presence of selective PPAR-ß antagonist (GSK0660) or PPAR-γ antagonist (GW9662), the inhibition of magnolol on collagen-induced platelet aggregation and intracellular Ca(2+) mobilization was significantly reversed. Moreover, magnolol-mediated up-regulation of NO/cyclic GMP/PKG pathway and Akt phosphorylation leading to increase of eNOS activity were markedly abolished by blocking PPAR-ß/γ activity. Additionally, magnolol significantly inhibited collagen-induced PKCα activation through a PPAR-ß/γ and PKCα interaction manner. The arachidonic acid (AA) or collagen-induced thromboxane B(2) formation and elevation of COX-1 activity caused by AA were also markedly attenuated by magnolol. However, these above effects of magnolol on platelet responses were strongly reduced by simultaneous addition of GSK0660 or GW9662, suggesting that PPAR-ß/γ-mediated processes may account for magnolol-regulated antiplatelet mechanisms. Similarly, administration of PPAR-ß/γ antagonists remarkably abolished the actions of magnolol in preventing platelet plug formation and prolonging bleeding time in mice. Taken together, we demonstrate for the first time that the antiplatelet and anti-thrombotic activities of magnolol are modulated by up-regulation of PPAR-ß/γ-dependent pathways.

Inhibitory effect of α-lipoic acid on platelet aggregation is mediated by PPARs.

Peroxisome proliferator-activated receptors (PPARs) isoforms (α, ß/δ, and γ are present in human platelets, and activation of PPARs inhibits platelet aggregation. α-Lipoic acid (ALA), occurring naturally in human food, has been reported to exhibit an antiplatelet activity. However, the mechanisms underlying ALA-mediated inhibition of platelet aggregation remain unknown. The aim of this study was to investigate whether the antiplatelet activity of ALA is mediated by PPARs. ALA itself significantly induced PPARα/γ activation in platelets and increased intracellular amounts of PPARα/γ by blocking PPARα/γ secretion from arachidonic acid (AA)-activated platelets. Moreover, ALA significantly inhibited AA-induced platelet aggregation, Ca(2+) mobilization, and cyclooxygenase-1 (COX-1) activity, but increased cyclic AMP production in rabbit washed platelets. Importantly, ALA also enhanced interaction of PPARα/γ with protein kinase Cα (PKCα) and COX-1 accompanied by an inhibition of PKCα activity in resting and AA-activated platelets. However, the above effects of ALA on platelets were markedly reversed by simultaneous addition of selective PPARα antagonist (GW6471) or PPARγ antagonist (GW9662). Taken together, the present study provides a novel mechanism by which ALA inhibition of platelet aggregation is mediated by PPARα/γ-dependent processes, which involve interaction with PKCα and COX-1, increase of cyclic AMP formation, and inhibition of intracellular Ca(2+) mobilization.

Antiplatelet activity of alpha-lipoic acid.

Alpha-lipoic acid (ALA) is often used as a dietary supplement to prevent and treat chronic diseases associated with excessive oxidative stress. The aim of this study was to investigate the mechanisms of the antiplatelet activity of ALA. ALA significantly inhibited collagen-induced platelet aggregation, thromboxane B(2) (TXB(2)) formation, Ca(2+) mobilization, and protein kinase Calpha (PKCalpha) activation, but ALA itself increased cyclic AMP formation in rabbit washed platelets. However, the effects of ALA on the above platelet responses were markedly reversed by the addition of 2'5'-ddAdo, an adenylate cyclase inhibitor. Additionally, increased reactive oxygen species (ROS) formation and cyclooxygenase-1 activity stimulated by arachidonic acid were inhibited by ALA. In conclusion, we demonstrated that ALA possesses an antiplatelet activity, which may be associated with an elevation of cyclic AMP formation, involving subsequent inhibition of TXA(2), Ca(2+) mobilization, and PKCalpha-mediated pathways. Moreover, inhibition of ROS formation and increase of platelet membrane fluidity may also involve its actions.