Objective
Increased protein phosphatase magnesium‐dependent 1A (PPM1A) levels in patients with ankylosing spondylitis regulate osteoblast differentiation in bony ankylosis; however, the potential ...mechanisms that regulate osteoclast differentiation in relation to abnormal bone formation remain unclear. This study was undertaken to investigate the relationship of PPM1A to osteoclast differentiation by generating conditional gene‐knockout (PPM1Afl/fl;LysM‐Cre) mice and evaluating their bone phenotype.
Methods
The bone phenotypes of LysM‐Cre mice (n = 6) and PPM1Afl/fl;LysM‐Cre mice (n = 6) were assessed by micro–computed tomography. Osteoclast differentiation was induced by culturing bone marrow–derived macrophages in the presence of RANKL and macrophage colony‐stimulating factor (M‐CSF), and was evaluated by counting tartrate‐resistant acid phosphatase–positive multinucleated cells. Levels of messenger RNA for PPM1A, RANK, and osteoclast‐specific genes were examined by real‐time quantitative polymerase chain reaction, and protein levels were determined by Western blotting. Surface RANK expression was analyzed by fluorescence flow cytometry.
Results
The PPM1Afl/fl;LysM‐Cre mice displayed reduced bone mass (P < 0.001) and increased osteoclast differentiation (P < 0.001) and osteoclast‐specific gene expression (P < 0.05) compared with their LysM‐Cre littermates. Mechanistically, reduced PPM1A function in osteoclast precursors in PPM1Afl/fl;LysM‐Cre mice induced osteoclast lineage commitment by up‐regulating RANK expression (P < 0.01) via p38 MAPK activation in response to M‐CSF. PPM1A expression in macrophages was decreased by Toll‐like receptor 4 activation (P < 0.05). The Ankylosing Spondylitis Disease Activity Score was negatively correlated with the expression of PPM1A in peripheral blood mononuclear cells from patients with axial spondyloarthritis (SpA) (γ = −0.7072, P < 0.0001).
Conclusion
The loss of PPM1A function in osteoclast precursors driven by inflammatory signals contributes to osteoclast lineage commitment and differentiation by elevating RANK expression, reflecting a potential role of PPM1A in dynamic bone metabolism in axial SpA.
Coupling is the process that links bone resorption to bone formation in a temporally and spatially coordinated manner within the remodeling cycle. Several lines of evidence point to the critical ...roles of osteoclast-derived coupling factors in the regulation of osteoblast performance. Here, we used a fractionated secretomic approach and identified the axon-guidance molecule SLIT3 as a clastokine that stimulated osteoblast migration and proliferation by activating β-catenin. SLIT3 also inhibited bone resorption by suppressing osteoclast differentiation in an autocrine manner. Mice deficient in Slit3 or its receptor, Robo1, exhibited osteopenic phenotypes due to a decrease in bone formation and increase in bone resorption. Mice lacking Slit3 specifically in osteoclasts had low bone mass, whereas mice with either neuron-specific Slit3 deletion or osteoblast-specific Slit3 deletion had normal bone mass, thereby indicating the importance of SLIT3 as a local determinant of bone metabolism. In postmenopausal women, higher circulating SLIT3 levels were associated with increased bone mass. Notably, injection of a truncated recombinant SLIT3 markedly rescued bone loss after an ovariectomy. Thus, these results indicate that SLIT3 plays an osteoprotective role by synchronously stimulating bone formation and inhibiting bone resorption, making it a potential therapeutic target for metabolic bone diseases.
Clusterin (CLU) is a heterodimeric glycoprotein involved in a range of biological processes. We investigated the function of CLU as a novel regulator of adipogenesis. CLU expression increased during ...3T3‐L1 preadipocyte differentiation. CLU overexpression promoted adipogenic differentiation of preadipocytes and increased the mRNA levels of adipogenic markers including peroxisome proliferator‐activated receptor γ (Pparg) and CCAAT enhancer‐binding protein α (Cebpa). Conversely, knockdown of CLU attenuated adipogenesis and reduced transcript levels of Pparg and Cebpa. However, the promoter activities of both the Pparg and the Cebpa gene were not affected by alteration of CLU expression on its own. Additionally, the protein level of Krüppel‐like factor 5 (KLF5), an upstream transcription factor of Pparg and Cebpa involved in adipogenic differentiation, was upregulated by CLU overexpression, although the mRNA level of Klf5 was not altered by changes in the expression level of CLU. Cycloheximide chase assay showed that the increased level of KLF5 by CLU overexpression was due to decreased degradation of KLF5 protein. Interestingly, CLU increased the stability of KLF5 by decreasing KLF5 ubiquitination. CLU inhibited the interaction between KLF5 and F‐box/WD repeat‐containing protein 7, which is an E3 ubiquitin ligase that targets KLF5. The adipogenic role of CLU was also addressed in mesenchymal stem cells (MSCs) and Clu−/− mouse embryonic fibroblasts (MEFs). Furthermore, CLU enhanced KLF5‐mediated transcriptional activation of both the Cebpa and the Pparg promoter. Taken together, these results suggest that CLU is a novel regulator of adipocyte differentiation by modulating the protein stability of the adipogenic transcription factor KLF5.
The tumor suppressor Smad4, a key mediator of the TGF‐β/BMP pathways, is essential for development and tissue homeostasis. Phosphorylation of Smad4 in its linker region catalyzed by the ...mitogen‐activated protein kinase (MAPK) plays a pivotal role in regulating its transcriptional activity and stability. In contrast, roles of Smad4 dephosphorylation as a control mechanism of TGF‐β/BMP signaling and the phosphatases responsible for its dephosphorylation remain so far elusive. Here, we identify Wip1 as a Smad4 phosphatase. Wip1 selectively binds and dephosphorylates Smad4 at Thr277, a key MAPK phosphorylation site, thereby regulating its nuclear accumulation and half‐life. In Xenopus embryos, Wip1 limits mesoderm formation and favors neural induction by inhibiting TGF‐β/BMP signals. Wip1 restrains TGF‐β‐induced growth arrest, migration, and invasion in human cells and enhances the tumorigenicity of cancer cells by repressing the antimitogenic activity of Smad4. We propose that Wip1‐dependent dephosphorylation of Smad4 is critical for the regulation of TGF‐β signaling.
Synopsis
Phosphorylation of Smad4, a central mediator of TGF‐β signaling, plays a pivotal role in controlling its activity and stability. Here, Wip1‐mediated dephosphorylation of Smad4 in its linker region is identified as a novel regulatory mechanism of TGF‐β signaling.
Wip1 negatively controls gene responses induced by TGF‐β/Activin/Nodal or BMP signals.
Wip1 directly dephosphorylates Smad4 at Thr277 in its linker region, regulating its nuclear retention and half‐life.
Wip1 restricts mesoderm formation and favours neural differentiation by dephosphorylating Smad4 in Xenopus.
Wip1 restrains migration of cancer cells, but promotes their tumourigenicity by down‐regulating Smad4 activity.
Phosphorylation of Smad4, a central mediator of TGF‐β signaling, plays a pivotal role in controlling its activity and stability. Here, Wip1‐mediated dephosphorylation of Smad4 in its linker region is identified as a novel regulatory mechanism of TGF‐β signaling.
The endoplasmic reticulum (ER) is a subcellular organelle essential for cellular homeostasis. Perturbation of ER functions due to various conditions can induce apoptosis. Chronic ER stress has been ...implicated in a wide range of diseases, including autosomal dominant retinitis pigmentosa (ADRP), which is characterized by age-dependent retinal degeneration caused by mutant rhodopsin alleles. However, the signaling pathways that mediate apoptosis in response to ER stress remain poorly understood. In this study, we performed an unbiased in vivo RNAi screen with a Drosophila ADRP model and found that Wg/Wnt1 mediated apoptosis. Subsequent transcriptome analysis revealed that ER stress-associated serine protease (Erasp), which has been predicted to show serine-type endopeptidase activity, was a downstream target of Wg/Wnt1 during ER stress. Furthermore, knocking down Erasp via RNAi suppressed apoptosis induced by mutant rhodopsin-1 (Rh-1
) toxicity, alleviating retinal degeneration in the Drosophila ADRP model. In contrast, overexpression of Erasp resulted in enhanced caspase activity in Drosophila S2 cells treated with apoptotic inducers and the stabilization of the initiator caspase Dronc (Death regulator Nedd2-like caspase) by stimulating DIAP1 (Drosophila inhibitor of apoptosis protein 1) degradation. These findings helped identify a novel cell death signaling pathway involved in retinal degeneration in an autosomal dominant retinitis pigmentosa model.
Although autophagy plays a role in melanogenesis by regulating melanosome degradation and biogenesis in melanocytes, a detailed understanding of the regulatory functions of autophagy factors is ...lacking. Here, we report a mechanistic link between microtubule-associated protein light chain 3 (LC3) activation and melanogenesis. We observed high expression of LC3 in melanosome-associated pigment-rich melanocytic nevi of sun-exposed skin, as indicated by patterns of melanosomal protein MART1 expression. Rapamycin-induced autophagy significantly increased the melanin index, tyrosinase activity and expression of several proteins linked to melanosome biogenesis, including microphthalmia transcription factor (MITF), pre-melanosome protein and tyrosinase, in Melan-a melanocytes. siRNA-mediated knockdown of LC3, but not beclin-1 or ATG5, decreased melanin content and tyrosinase activity. LC3 knockdown also markedly inhibited MITF expression and subsequent rapamycin-induced melanosome formation. More importantly, LC3 knockdown suppressed α-MSH-mediated melanogenesis by attenuating cAMP response element-binding protein (CREB) phosphorylation and MITF expression in Melan-a cells via decreased extracellular signal-regulated kinase (ERK) activity. Overexpression of constitutively active ERK reversed the effect of LC3 knockdown on CREB phosphorylation and MITF expression. These findings demonstrate that LC3 contributes to melanogenesis by increasing ERK-dependent MITF expression, thereby providing a mechanistic insight into the signaling network that links autophagy to melanogenesis.
Patients with fibro-calcific aortic valve disease (FCAVD) have lipid depositions in their aortic valve that engender a proinflammatory impetus toward fibrosis and calcification and ultimately valve ...leaflet stenosis. Although the lipoprotein(a)-autotaxin (ATX)-lysophosphatidic acid axis has been suggested as a potential therapeutic target to prevent the development of FCAVD, supportive evidence using ATX inhibitors is lacking. We here evaluated the therapeutic potency of an ATX inhibitor to attenuate valvular calcification in the FCAVD animal models.
ATX level and activity in healthy participants and patients with FCAVD were analyzed using a bioinformatics approach using the Gene Expression Omnibus datasets, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and western blotting. To evaluate the efficacy of ATX inhibitor, interleukin-1 receptor antagonist-deficient (Il1rn
mice and cholesterol-enriched diet-induced rabbits were used as the FCAVD models, and primary human valvular interstitial cells (VICs) from patients with calcification were employed.
The global gene expression profiles of the aortic valve tissue of patients with severe FCAVD demonstrated that ATX gene expression was significantly upregulated and correlated with lipid retention (r = 0.96) or fibro-calcific remodeling-related genes (r = 0.77) in comparison to age-matched non-FCAVD controls. Orally available ATX inhibitor, BBT-877, markedly ameliorated the osteogenic differentiation and further mineralization of primary human VICs in vitro. Additionally, ATX inhibition significantly attenuated fibrosis-related factors' production, with a detectable reduction of osteogenesis-related factors, in human VICs. Mechanistically, ATX inhibitor prohibited fibrotic changes in human VICs via both canonical and non-canonical TGF-β signaling, and subsequent induction of CTGF, a key factor in tissue fibrosis. In the in vivo FCAVD model system, ATX inhibitor exposure markedly reduced calcific lesion formation in interleukin-1 receptor antagonist-deficient mice (Il1rn
, P = 0.0210). This inhibition ameliorated the rate of change in the aortic valve area (P = 0.0287) and mean pressure gradient (P = 0.0249) in the FCAVD rabbit model. Moreover, transaortic maximal velocity (Vmax) was diminished with ATX inhibitor administration (mean Vmax = 1.082) compared to vehicle control (mean Vmax = 1.508, P = 0.0221). Importantly, ATX inhibitor administration suppressed the effects of a high-cholesterol diet and vitamin D2-driven fibrosis, in association with a reduction in macrophage infiltration and calcific deposition, in the aortic valves of this rabbit model.
ATX inhibition attenuates the development of FCAVD while protecting against fibrosis and calcification in VICs, suggesting the potential of using ATX inhibitors to treat FCAVD.
The CRISPR-Cas9 system has significantly advanced regenerative medicine research by enabling genome editing in stem cells. Due to their desirable properties, mesenchymal stem cells (MSCs) have ...recently emerged as highly promising therapeutic agents, which properties include differentiation ability and cytokine production. While CRISPR-Cas9 technology is applied to develop MSC-based therapeutics, MSCs exhibit inefficient genome editing, and susceptibility to plasmid DNA. In this study, we compared and optimized plasmid DNA and RNP approaches for efficient genome engineering in MSCs. The RNP-mediated approach enabled genome editing with high indel frequency and low cytotoxicity in MSCs. By utilizing Cas9 RNPs, we successfully generated B2M-knockout MSCs, which reduced T-cell differentiation, and improved MSC survival. Furthermore, this approach enhanced the immunomodulatory effect of IFN-r priming. These findings indicate that the RNP-mediated engineering of MSC genomes can achieve high efficiency, and engineered MSCs offer potential as a promising therapeutic strategy. BMB Reports 2024; 57(1): 60-65.
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