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1.
Physiol Genomics ; 2024 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-39311838

RESUMO

The extracellular isoform of superoxide dismutase (SOD3) is decreased in patients and animals with pulmonary hypertension (PH). The human R213G single nucleotide polymorphism (SNP) in SOD3 causes its release from tissue extracellular matrix (ECM) into extracellular fluids, without modulating enzyme activity, increasing cardiovascular disease risk in humans and exacerbating chronic hypoxic PH in mice. Given the importance of interstitial macrophages (IM) to PH pathogenesis, this study aimed to determine whether R213G SOD3 increases IM accumulation and alters IM reprogramming in response to hypoxia. R213G mice and wild-type (WT) controls were exposed to hypobaric hypoxia for 4 or 14 days compared to normoxia. Flow cytometry demonstrated a transient increase in IMs at day 4 in both strains. Contrary to our hypothesis, the R213G SNP did not augment IM accumulation. To determine strain differences in the IM reprogramming response to hypoxia, we performed RNAsequencing on IMs isolated at each time point. We found that IMs from R213G mice exposed to hypoxia activated ECM-related pathways and a combination of alternative macrophage and proinflammatory signaling. Furthermore, when compared to WT responses, IMs from R213G mice lacked metabolic remodeling and demonstrated a blunted anti-inflammatory response between the early (day 4) and later (day 14) time points. We confirmed metabolic responses using Agilent Seahorse assays whereby WT, but not R213G, IMs upregulated glycolysis at day 4 that returned to baseline at day 14. Finally, we identify differential regulation of several redox-sensitive upstream regulators that could be investigated in future studies.

2.
Antioxidants (Basel) ; 13(8)2024 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-39199221

RESUMO

Pulmonary hypertension (PH) is a progressive disease marked by pulmonary vascular remodeling and right ventricular failure. Inflammation and oxidative stress are critical in PH pathogenesis, with early pulmonary vascular inflammation preceding vascular remodeling. Extracellular superoxide dismutase (EC-SOD), a key vascular antioxidant enzyme, mitigates oxidative stress and protects against inflammation and fibrosis in diverse lung and vascular disease models. This study utilizes a murine hypobaric hypoxia model to investigate the role of lung EC-SOD on hypoxia-induced platelet activation and platelet lung accumulation, a critical factor in PH-related inflammation. We found that lung EC-SOD overexpression blocked hypoxia-induced platelet activation and platelet accumulation in the lung. Though lung EC-SOD overexpression increased lung EC-SOD content, it did not impact plasma extracellular SOD activity. However, ex vivo, exogenous extracellular SOD treatment specifically blunted convulxin-induced platelet activation but did not blunt platelet activation with thrombin or ADP. Our data identify platelets as a novel target of EC-SOD in response to hypoxia, providing a foundation to advance the understanding of dysregulated redox signaling and platelet activation in PH and other chronic hypoxic lung diseases.

3.
Bone ; 172: 116759, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37044359

RESUMO

Studies using kinase inhibitors have shown that the protein kinase D (PRKD) family of serine/threonine kinases are required for formation and function of osteoclasts in culture. However, the involvement of individual protein kinase D genes and their in vivo significance to skeletal dynamics remains unclear. In the current study we present data indicating that protein kinase D3 is the primary form of PRKD expressed in osteoclasts. We hypothesized that loss of PRKD3 would impair osteoclast formation, thereby decreasing bone resorption and increasing bone mass. Conditional knockout (cKO) of Prkd3 using a murine Cre/Lox system driven by cFms-Cre revealed that its loss in osteoclast-lineage cells reduced osteoclast differentiation and resorptive function in culture. Examination of the Prkd3 cKO mice showed that bone parameters were unaffected in the femur at 4 weeks of age, but consistent with our hypothesis, Prkd3 conditional knockout resulted in 18 % increased trabecular bone mass in male mice at 12 weeks and a similar increase at 6 months. These effects were not observed in female mice. As a further test of our hypothesis, we asked if Prkd3 cKO could protect against bone loss in a ligature-induced periodontal disease model but did not see any reduction in bone destruction in this system. Together, our data indicate that PRKD3 promotes osteoclastogenesis both in vitro and in vivo.


Assuntos
Reabsorção Óssea , Osteólise , Masculino , Feminino , Camundongos , Animais , Osteoclastos/metabolismo , Osso Esponjoso/metabolismo , Reabsorção Óssea/metabolismo , Osteogênese , Osteólise/metabolismo , Camundongos Knockout , Diferenciação Celular/genética
4.
Bone ; 159: 116393, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35318161

RESUMO

Protein kinase D (PRKD) family kinases are required for formation and function of osteoclasts. However, the substrates of PRKD in osteoclasts are unknown. To identify PRKD-dependent protein phosphorylation in osteoclasts, we performed a quantitative LC-MS/MS phosphoproteomics screen for proteins showing differential phosphorylation in osteoclasts after treatment with the PRKD inhibitor CRT0066101. We identified 757 phosphopeptides showing significant changes following PRKD inhibition. Among the changes, we found a group of 13 proteins showing decreased phosphorylation at PRKD consensus phosphorylation motifs. This group includes histone deacetylase 5 (HDAC5), which is a previously validated PRKD target. Considering this known interaction, work suggesting HDACs may be important regulators of osteoclasts, and studies suggesting potential functional redundancy between HDACs, we further investigated the relationship between PRKD and class IIa HDACs in osteoclasts. We confirmed that CRT0066101 inhibits phosphorylation of endogenous HDAC5 and to a lesser extent HDAC4, whereas HDAC7 phosphorylation was not affected. Osteoclast cultures from Hdac5 global knockout mice displayed impaired differentiation and reduced ability to resorb bone, while conditional knockout of Hdac4 in osteoclasts showed no phenotype in vitro or in vivo. The inhibitory effect of CRT0066101 was reduced in Hdac5 KO osteoclasts. Together these data indicate that the PRKD/HDAC5 axis contributes to osteoclast formation in vitro and suggest that this pathway may contribute to regulation of skeletal dynamics in vivo.


Assuntos
Osteoclastos , Espectrometria de Massas em Tandem , Animais , Cromatografia Líquida , Histona Desacetilases/metabolismo , Camundongos , Osteoclastos/metabolismo , Fosforilação , Proteína Quinase C
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