Soluble Klotho causes hypomineralization in Klotho-deficient mice

The type I transmembrane protein αKlotho (Klotho) serves as a coreceptor for the phosphaturic hormone fibroblast growth factor 23 (FGF23) in kidney, while a truncated form of Klotho (soluble Klotho, sKL) is thought to exhibit multiple activities, including acting as a hormone, but whose mode(s) of action in different organ systems remains to be fully elucidated. FGF23 is expressed primarily in osteoblasts/osteocytes and aberrantly high levels in the circulation acting via signaling through an FGF receptor (FGFR)-Klotho coreceptor complex cause renal phosphate wasting and osteomalacia. We assessed the effects of exogenously added sKL on osteoblasts and bone using Klotho-deficient (kl/kl) mice and cell and organ cultures. sKL induced FGF23 signaling in bone and exacerbated the hypomineralization without exacerbating the hyperphosphatemia, hypercalcemia and hypervitaminosis D in kl/kl mice. The same effects were seen in rodent bone models in vitro, in which we also detected formation of a sKL complex with FGF23-FGFR and decreased Phex (gene responsible for X-linked hypophosphatemic rickets (XLH)/osteomalacia) expression. Further, sKL-FGF23-dependent hypomineralization in vitro was rescued by soluble PHEX. These data suggest that exogenously added sKL directly participates in FGF23 signaling in bone and that PHEX is a downstream effector of the sKL-FGF23-FGFR axis in bone.

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Pleiotropic Actions of FGF23

Toxicologic Pathology ◽

10.1177/0192623317737469 ◽

2017 ◽

Vol 45 (7) ◽

pp. 904-910 ◽

Cited By ~ 20

Author(s):

Reinhold G. Erben

Keyword(s):

Vitamin D ◽

Fibroblast Growth Factor 23 ◽

Renal Tubules ◽

Hormone Production ◽

Membrane Expression ◽

Fgf Receptor ◽

Local Inhibition ◽

Organ Systems ◽

Key Enzyme ◽

Renal Tubular

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone, mainly produced by osteoblasts and osteocytes in response to increased extracellular phosphate and circulating vitamin D hormone. Endocrine FGF23 signaling requires co-expression of the ubiquitously expressed FGF receptor 1 (FGFR1) and the co-receptor α-Klotho (Klotho). In proximal renal tubules, FGF23 suppresses the membrane expression of the sodium–phosphate cotransporters Npt2a and Npt2c which mediate urinary reabsorption of filtered phosphate. In addition, FGF23 suppresses proximal tubular expression of 1α-hydroxylase, the key enzyme responsible for vitamin D hormone production. In distal renal tubules, FGF23 signaling activates with-no-lysine kinase 4, leading to increased renal tubular reabsorption of calcium and sodium. Therefore, FGF23 is not only a phosphaturic but also a calcium- and sodium-conserving hormone, a finding that may have important implications for the pathophysiology of chronic kidney disease. Besides these endocrine, Klotho-dependent functions of FGF23, FGF23 is also an auto-/paracrine suppressor of tissue-nonspecific alkaline phosphatase transcription via Klotho-independent FGFR3 signaling, leading to local inhibition of mineralization through accumulation of pyrophosphate. In addition, FGF23 may target the heart via an FGFR4-mediated Klotho-independent signaling cascade. Taken together, there is emerging evidence that FGF23 is a pleiotropic hormone, linking bone with several other organ systems.

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STAT2-Dependent Immune Responses Ensure Host Survival despite the Presence of a Potent Viral Antagonist

Journal of Virology ◽

10.1128/jvi.00296-18 ◽

2018 ◽

Vol 92 (14) ◽

Cited By ~ 10

Author(s):

Vu Thuy Khanh Le-Trilling ◽

Kerstin Wohlgemuth ◽

Meike U. Rückborn ◽

Andreja Jagnjic ◽

Fabienne Maaßen ◽

...

Keyword(s):

Immune Responses ◽

Viral Replication ◽

Mutual Influence ◽

Type I ◽

Mcmv Infection ◽

General Importance ◽

Interferon Antagonism ◽

Deficient Mice

ABSTRACTA pathogen encounter induces interferons, which signal via Janus kinases and STAT transcription factors to establish an antiviral state. However, the host and pathogens are situated in a continuous arms race which shapes host evolution toward optimized immune responses and the pathogens toward enhanced immune-evasive properties. Mouse cytomegalovirus (MCMV) counteracts interferon responses by pM27-mediated degradation of STAT2, which directly affects the signaling of type I as well as type III interferons. Using MCMV mutants lackingM27and mice lacking STAT2, we studied the opposing relationship between antiviral activities and viral antagonism in a natural host-pathogen pairin vitroandin vivo. In contrast to wild-type (wt) MCMV, ΔM27 mutant MCMV was efficiently cleared from all organs within a few days in BALB/c, C57BL/6, and 129 mice, highlighting the general importance of STAT2 antagonism for MCMV replication. Despite this effective and relevant STAT2 antagonism, wt and STAT2-deficient mice exhibited fundamentally different susceptibilities to MCMV infections. MCMV replication was increased in all assessed organs (e.g., liver, spleen, lungs, and salivary glands) of STAT2-deficient mice, resulting in mortality during the first week after infection. Taken together, the results of our study reveal the importance of cytomegaloviral interferon antagonism for viral replication as well as a pivotal role of the remaining STAT2 activity for host survival. This mutual influence establishes a stable evolutionary standoff situation with fatal consequences when the equilibrium is disturbed.IMPORTANCEThe host limits viral replication by the use of interferons (IFNs), which signal via STAT proteins. Several viruses evolved antagonists targeting STATs to antagonize IFNs (e.g., cytomegaloviruses, Zika virus, dengue virus, and several paramyxoviruses). We analyzed infections caused by MCMV expressing or lacking the STAT2 antagonist pM27 in STAT2-deficient and control mice to evaluate its importance for the host and the virusin vitroandin vivo. The inability to counteract STAT2 directly translates into exaggerated IFN susceptibilityin vitroand pronounced attenuationin vivo. Thus, the antiviral activity mediated by IFNs via STAT2-dependent signaling drove the development of a potent MCMV-encoded STAT2 antagonist which became indispensable for efficient virus replication and spread to organs required for dissemination. Despite this clear impact of viral STAT2 antagonism, the host critically required the remaining STAT2 activity to prevent overt disease and mortality upon MCMV infection. Our findings highlight a remarkably delicate balance between host and virus.

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Design, Synthesis and Biological Evaluation of (2′,5′ and 3′5′-Linked) cGAMP Analogs that Activate Stimulator of Interferon Genes (STING)

Molecules ◽

10.3390/molecules25225285 ◽

2020 ◽

Vol 25 (22) ◽

pp. 5285

Author(s):

Xin Xie ◽

Junyi Liu ◽

Xiaowei Wang

Keyword(s):

Transmembrane Protein ◽

Biological Evaluation ◽

Type I ◽

Vaccine Adjuvants ◽

One Pot ◽

Design Synthesis ◽

Cellular Assays ◽

Type I Ifns ◽

Sting Pathway

Stimulator of interferon genes (STING) is an endoplasmic reticulum adaptor transmembrane protein that plays a pivotal role in innate immune system. STING agonists, such as endogenous cyclic dinucleotide (CDN) cyclic GMP-AMP (cGAMP), have been used in diverse clinical research for immunogenic tumor clearance, antiviral treatments and vaccine adjuvants. CDNs containing noncanonical mixed 3′-5′ and 2′-5′ phosphodiester linkages show higher potency in the activation of the STING pathway. In this study, a series of 2′3′-CDNs were designed and synthesized through a modified one-pot strategy. We then established a surface plasmon resonance (SPR)-based binding assay to quantify the binding affinities of synthesized CDNs for human STING, which requested a minuscule amount of sample without any pretreatment. Using this assay, we identified compound 8d (KD = 0.038 μM), a novel CDN that showed higher binding affinity with hSTING than cGAMP (KD = 0.543 μM). Cellular assays confirmed that 8d could trigger the expression of type I IFNs and other proinflammatory cytokines more robust than cGAMP. 8d also exhibited more resistant than cGAMP to enzymatic cleavage in vitro, indicating the successful improvement in drug availability. These findings provide guidelines for the design and structural optimization of CDNs as STING agonists.

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Contractile properties of EDL and soleus muscles of myostatin-deficient mice

Journal of Applied Physiology ◽

10.1152/japplphysiol.00126.2006 ◽

2006 ◽

Vol 101 (3) ◽

pp. 898-905 ◽

Cited By ~ 95

Author(s):

Christopher L. Mendias ◽

James E. Marcin ◽

Daniel R. Calerdon ◽

John A. Faulkner

Keyword(s):

Type I Collagen ◽

Contractile Properties ◽

Negative Regulator ◽

Type I ◽

Lengthening Contractions ◽

Tetanic Force ◽

The Difference ◽

The Impact ◽

Deficient Mice

Myostatin is a negative regulator of muscle mass. The impact of myostatin deficiency on the contractile properties of healthy muscles has not been determined. We hypothesized that myostatin deficiency would increase the maximum tetanic force (Po), but decrease the specific Po(sPo) of muscles and increase the susceptibility to contraction-induced injury. The in vitro contractile properties of extensor digitorum longus (EDL) and soleus muscles from wild-type ( MSTN+/+), heterozygous-null ( MSTN+/−), and homozygous-null ( MSTN−/−) adult male mice were determined. For EDL muscles, the Poof both MSTN+/−and MSTN−/−mice were greater than the Poof MSTN+/+mice. For soleus muscles, the Poof MSTN−/−mice was greater than that of MSTN+/+mice. The sPoof EDL muscles of MSTN−/−mice was less than that of MSTN+/+mice. For soleus muscles, however, no difference in sPowas observed. Following two lengthening contractions, EDL muscles from MSTN−/−mice had a greater force deficit than that of MSTN+/+or MSTN+/−mice, whereas no differences were observed for the force deficits of soleus muscles. Myostatin-deficient EDL muscles had less hydroxyproline, and myostatin directly increased type I collagen mRNA expression and protein content. The difference in the response of EDL and soleus muscles to myostatin may arise from differences in the levels of a myostatin receptor, activin type IIB. Compared with the soleus, the amount of activin type IIB receptor was approximately twofold greater in EDL muscles. The results support a significant role for myostatin not only in the mass of muscles but also in the contractility and the composition of the extracellular matrix of muscles.

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Gab2 deficiency prevents Flt3-ITD driven acute myeloid leukemia in vivo

Leukemia ◽

10.1038/s41375-021-01490-0 ◽

2021 ◽

Author(s):

Corinna Spohr ◽

Teresa Poggio ◽

Geoffroy Andrieux ◽

Katharina Schönberger ◽

Nina Cabezas-Wallscheid ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Tyrosine Kinases ◽

Myeloid Leukemia ◽

Downstream Effector ◽

Flt3 Inhibitor ◽

Genes Encoding ◽

Deficient Mice ◽

Acute Myeloid

AbstractInternal tandem duplications (ITD) of the FMS-like tyrosine kinase 3 (FLT3) predict poor prognosis in acute myeloid leukemia (AML) and often co-exist with inactivating DNMT3A mutations. In vitro studies implicated Grb2-associated binder 2 (GAB2) as FLT3-ITD effector. Utilizing a Flt3-ITD knock-in, Dnmt3a haploinsufficient mouse model, we demonstrate that Gab2 is essential for the development of Flt3-ITD driven AML in vivo, as Gab2 deficient mice displayed prolonged survival, presented with attenuated liver and spleen pathology and reduced blast counts. Furthermore, leukemic bone marrow from Gab2 deficient mice exhibited reduced colony-forming unit capacity and increased FLT3 inhibitor sensitivity. Using transcriptomics, we identify the genes encoding for Axl and the Ret co-receptor Gfra2 as targets of the Flt3-ITD/Gab2/Stat5 axis. We propose a pathomechanism in which Gab2 increases signaling of these receptors by inducing their expression and by serving as downstream effector. Thereby, Gab2 promotes AML aggressiveness and drug resistance as it incorporates these receptor tyrosine kinases into the Flt3-ITD signaling network. Consequently, our data identify GAB2 as a promising biomarker and therapeutic target in human AML.

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TRIM35 mediates protection against influenza infection by activating TRAF3 and degrading viral PB2

Protein & Cell ◽

10.1007/s13238-020-00734-6 ◽

2020 ◽

Vol 11 (12) ◽

pp. 894-914

Author(s):

Nan Sun ◽

Li Jiang ◽

Miaomiao Ye ◽

Yihan Wang ◽

Guangwen Wang ◽

...

Keyword(s):

Influenza A ◽

Viral Infections ◽

Influenza Infection ◽

Type I ◽

Subsequent Formation ◽

Antiviral Immune Response ◽

Signaling Complex ◽

Deficient Mice

AbstractTripartite motif (TRIM) family proteins are important effectors of innate immunity against viral infections. Here we identified TRIM35 as a regulator of TRAF3 activation. Deficiency in or inhibition of TRIM35 suppressed the production of type I interferon (IFN) in response to viral infection. Trim35-deficient mice were more susceptible to influenza A virus (IAV) infection than were wild-type mice. TRIM35 promoted the RIG-I-mediated signaling by catalyzing Lys63-linked polyubiquitination of TRAF3 and the subsequent formation of a signaling complex with VISA and TBK1. IAV PB2 polymerase countered the innate antiviral immune response by impeding the Lys63-linked polyubiquitination and activation of TRAF3. TRIM35 mediated Lys48-linked polyubiquitination and proteasomal degradation of IAV PB2, thereby antagonizing its suppression of TRAF3 activation. Our in vitro and in vivo findings thus reveal novel roles of TRIM35, through catalyzing Lys63- or Lys48-linked polyubiquitination, in RIG-I antiviral immunity and mechanism of defense against IAV infection.

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A Phase I, Multi-Dose, Dose Escalation Study of SGN-40 (anti-huCD40 mAb) in Patients with Refractory or Recurrent Multiple Myeloma.

Blood ◽

10.1182/blood.v104.11.2413.2413 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2413-2413

Author(s):

Mohamad A. Hussein ◽

Ruben Niesvizky ◽

Nikhil Munshi ◽

James C. Berenson ◽

Kenneth C. Anderson ◽

...

Keyword(s):

Multiple Myeloma ◽

Phase I ◽

Dose Escalation ◽

Transmembrane Protein ◽

M Protein ◽

Type I ◽

Antitumor Effects ◽

Grade 3 ◽

Dose Levels

Abstract CD40 is a type I transmembrane protein that upon binding to CD40 ligand regulates important biologic effects in the immune system. CD40 is also highly expressed on hematologic tumors, which has raised interest in the potential for its use as a tumor target for antibody-based cancer therapy. SGN-40 is a humanized monoclonal antibody that selectively binds to human CD40 and induces apoptosis and growth inhibition of a wide variety of B-cell derived cancer cell lines in vitro. Our preclinical work has confirmed the in vitro cytotoxicity of SGN-40 against human multiple myeloma (MM) cells via several mechanisms. These include induction of cytotoxic ligands of TNF superfamily; suppression of IL-6-induced proliferative and anti-apoptotic effects, as well as antibody-dependent cell-mediated cytotoxicity (Tai, et al, Cancer Research64, 2846–2852, April 15, 2004). Since ≥ 90% of MM cells express CD40, targeting CD40 using SGN-40 presents a potential novel treatment strategy. Based on these preclinical data, a phase I study is being conducted to define the toxicity profile, characterize the pharmacokinetics (PK), and evaluate antitumor effects of SGN-40 in patients with refractory or recurrent MM. Four weekly doses ranging from 0.5 to 16 mg/kg are planned to be administered to groups of at least three patients per cohort. Patients will be followed for up to 6 weeks post their last dose. Currently, a total of seven patients have been treated with SGN-40 at dose levels of 0.5 and 1.0 mg/kg. No grade 3 or 4 non-hematologic dose limiting toxicities have been observed. One patient experienced a transient Grade 3 decrease in hemoglobin. Decrease in CD19 positive B-cells were noted for patients treated at both dose levels. Changes in serum and urine M protein levels were measured to estimate potential anti-tumor effects of SGN-40. Of the seven patients evaluated, one patient at 0.5 mg/kg dose had stable disease, based on serum M protein, over the 10 week study period. Clinical evaluation with dose escalation of this agent continues and updated safety, PK and antitumor data will be presented.

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ASARM peptides: PHEX-dependent and -independent regulation of serum phosphate

AJP Renal Physiology ◽

10.1152/ajprenal.00304.2010 ◽

2011 ◽

Vol 300 (3) ◽

pp. F783-F791 ◽

Cited By ~ 34

Author(s):

Valentin David ◽

Aline Martin ◽

Anne-Marie Hedge ◽

Marc K. Drezner ◽

Peter S. N. Rowe

Keyword(s):

Vitamin D ◽

Fibroblast Growth Factor 23 ◽

Inverse Correlation ◽

Fractional Excretion ◽

Serum Phosphate ◽

Hypophosphatemic Rickets ◽

Separate Experiment ◽

Constitutive Overexpression ◽

Fractional Excretion Of Phosphate

Increased acidic serine aspartate-rich MEPE-associated motif (ASARM) peptides cause mineralization defects in X-linked hypophosphatemic rickets mice (HYP) and “directly” inhibit renal phosphate uptake in vitro. However, ASARM peptides also bind to phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and are a physiological substrate for this bone-expressed, phosphate-regulating enzyme. We therefore tested the hypothesis that circulating ASARM peptides also “indirectly” contribute to a bone-renal PHEX-dependent hypophosphatemia in normal mice. Male mice ( n = 5; 12 wk) were fed for 8 wk with a normal phosphorus and vitamin D3 diet (1% Pi diet) or a reduced phosphorus and vitamin D3 diet (0.1% Pi diet). For the final 4 wk, transplantation of mini-osmotic pumps supplied a continuous infusion of either ASARM peptide (5 mg·day−1·kg−1) or vehicle. HYP, autosomal recessive hypophosphatemic rickets (ARHR), and normal mice (no pumps or ASARM infusion; 0.4% Pi diet) were used in a separate experiment designed to measure and compare circulating ASARM peptides in disease and health. ASARM treatment decreased serum phosphate concentration and renal phosphate cotransporter (NPT2A) mRNA with the 1% Pi diet. This was accompanied by a twofold increase in serum ASARM and 1,25-dihydroxy vitamin D3 [1,25 (OH)2D3] levels without changes in parathyroid hormone. For both diets, ASARM-treated mice showed significant increases in serum fibroblast growth factor 23 (FGF23; +50%) and reduced serum osteocalcin (−30%) and osteopontin (−25%). Circulating ASARM peptides showed a significant inverse correlation with serum Pi and a significant positive correlation with fractional excretion of phosphate. We conclude that constitutive overexpression of ASARM peptides plays a “component” PHEX-independent part in the HYP and ARHR hypophosphatemia. In contrast, with wild-type mice, ASARM peptides likely play a bone PHEX-dependent role in renal phosphate regulation and FGF23 expression. They may also coordinate FGF23 expression by competitively modulating PHEX/DMP1 interactions and thus bone-renal mineral regulation.

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Fusion Potential of Human Osteoclasts In Vitro Reflects Age, Menopause, and In Vivo Bone Resorption Levels of Their Donors—A Possible Involvement of DC-STAMP

International Journal of Molecular Sciences ◽

10.3390/ijms21176368 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6368

Author(s):

Anaïs M. J. Møller ◽

Jean-Marie Delaissé ◽

Jacob B. Olesen ◽

Luisa M. Canto ◽

Silvia R. Rogatto ◽

...

Keyword(s):

Bone Resorption ◽

Type I Collagen ◽

Transmembrane Protein ◽

Type I ◽

Contributing Factors ◽

Expression Levels ◽

Procollagen Type ◽

Age Related

It is well established that multinucleation is central for osteoclastic bone resorption. However, our knowledge on the mechanisms regulating how many nuclei an osteoclast will have is limited. The objective of this study was to investigate donor-related variations in the fusion potential of in vitro-generated osteoclasts. Therefore, CD14+ monocytes were isolated from 49 healthy female donors. Donor demographics were compared to the in vivo bone biomarker levels and their monocytes’ ability to differentiate into osteoclasts, showing that: (1) C-terminal telopeptide of type I collagen (CTX) and procollagen type I N-terminal propeptide (PINP) levels increase with age, (2) the number of nuclei per osteoclast in vitro increases with age, and (3) there is a positive correlation between the number of nuclei per osteoclast in vitro and CTX levels in vivo. Furthermore, the expression levels of the gene encoding dendritic cell-specific transmembrane protein (DCSTAMP) of osteoclasts in vitro correlated positively with the number of nuclei per osteoclast, CTX levels in vivo, and donor age. Our results furthermore suggest that these changes in gene expression may be mediated through age-related changes in DNA methylation levels. We conclude that both intrinsic factors and age-induced increase in fusion potential of osteoclasts could be contributing factors for the enhanced bone resorption in vivo, possibly caused by increased expression levels of DCSTAMP.

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Identification of Small-Molecule Inhibitors of FGF23 Signaling via In Silico Hot Spot Prediction and Molecular Docking to α-Klotho

10.26434/chemrxiv.13083224.v2 ◽

2020 ◽

Author(s):

Shih-Hsien Liu ◽

Zhousheng Xiao ◽

Sambit K. Mishra ◽

Julie C. Mitchell ◽

Jeremy C. Smith ◽

...

Keyword(s):

Small Molecule ◽

In Silico ◽

Ternary Complex ◽

Hot Spot ◽

Fibroblast Growth Factor 23 ◽

Regular Function ◽

Fgf Receptor ◽

Zinc Database ◽

Extracellular Signal

Fibroblast growth factor 23 (FGF23) is a therapeutic target for treating hereditary and acquired hypophosphatemic disorders, such as X-linked hypophosphatemic (XLH) rickets and tumor-induced osteomalacia (TIO), respectively. FGF23-induced hypophosphatemia is mediated by signaling through a ternary complex formed by FGF23, FGF receptor (FGFR), and α-Klotho. Currently, disorders of excess FGF23 are treated with an FGF23-blocking antibody, Burosumab. Small-molecule drugs that disrupt protein:protein interactions necessary for the ternary complex formation offer an alternative to disrupt FGF23 signaling. In this study, the FGF23:α-Klotho interface was targeted to identify small-molecule protein:protein interaction inhibitors. We computationally identified “hot spots” in the FGF23:α-Klotho interface of the ternary complex and performed in silico docking of ~5.5 million compounds from the ZINC database to the interface region of α-Klotho from the ternary crystal structure. Following docking, 23 and 18 compounds were chosen based on the lowest binding free energies to α-Klotho and the largest number of contacts with Tyr433, a predicted hot spot, respectively. 5 compounds available were assessed experimentally by their FGF23-mediated extracellular signal-regulated kinase (ERK) activities in vitro, and two of these reduce activities significantly. Both these compounds have a favorable predicted binding affinity, but not a large number of contacts with the hot spot residues. ZINC12409120 was found experimentally to reduce FGF23-mediated ERK activities by 70% and have a half maximal inhibitory concentration (IC50) of 5.0 ± 0.23 uM. ZINC12409120 exhibits contacts with residues on KL1 and KL2 domains and on the linker between the two domains of α-Klotho in in silico binding poses, thereby possibly disrupting the regular function of α-Klotho and impeding FGF23 binding. ZINC12409120 is a candidate for lead optimization.

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