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1.
JBMR Plus ; 4(7): e10080, 2020 Jul.
Article in English | MEDLINE | ID: mdl-32666016

ABSTRACT

CSF-1 is a key factor in regulating bone remodeling; osteocytes express CSF-1 and its receptor. Viable osteocytes are essential for bone remodeling through cell-cell contact and secretion of factors that regulate osteoblasts and osteoclasts. Increased oxidative stress contributes to osteocyte death and correlates with bone loss during aging. The NADPH oxidase Nox4 is a major source of ROS in bone. CSF-1 decreases Nox4, suggesting that CSF-1 protects against oxidative stress. Here, we show that osteocyte apoptosis previously reported in our global CSF-1KO mice is associated with increased Nox4, as well as 4-HNE expression in osteocytes. Osteocytes isolated from CSF-1KO mice were less viable and showed increased intracellular ROS, elevated NADPH oxidase activity/Nox4 protein, activation of mTOR/S6K, and downstream apoptosis signals compared with WT osteocytes. Nox4 expression was also increased in CSF-1KO osteocytes and colocalized with MitoTracker Red in mitochondria. Notably, CSF-1 inhibited Nox4 expression and apoptosis cascade signals. In additional studies, shNox4 decreased these signals in CSF-1KO osteocytes, whereas overexpression of Nox4 in WT osteocytes activated the apoptosis pathway. To determine the role of CSF-1 in osteocytes, DMP1Cre-CSF-1cKO (CSF-1cKO) mice that lack CSF-1 in osteocytes/late osteoblasts were developed. Osteocyte defects in CSF-1cKO mice overlapped with those in CSF-1KO mice, including increased apoptosis, Nox4, and 4-HNE-expressing osteocytes. CSF-1cKO mice showed unbalanced cancellous bone remodeling with decreased bone formation and resorption. Continued exposure to high Nox4/ROS levels may further compromise bone formation and predispose to bone loss and skeletal fragility. Taken together, our findings suggest a novel link between CSF-1, Nox4-derived ROS, and osteocyte survival/function that is crucial for osteocyte-mediated bone remodeling. Results reveal new mechanisms by which CSF-1/oxidative stress regulate osteocyte homeostasis, which may lead to therapeutic strategies to improve skeletal health in aging. © 2018 American Society for Bone and Mineral Research.

2.
J Sport Rehabil ; 28(5): 432-437, 2019 Jul 01.
Article in English | MEDLINE | ID: mdl-29364042

ABSTRACT

Context: Numerous studies have reported kinematic data on baseball pitchers using three-dimensional (3D) motion analysis, but no studies to date have correlated this data with clinical outcome measures. Objective: To examine the relationship among Y-Balance Test-Lower Quarter (YBT-LQ) composite scores, musculoskeletal characteristics of the hip, and pitching kinematics in National Collegiate Athletic Association (NCAA) Division I baseball pitchers. Design: Cross-sectional. Setting: 3D motion analysis laboratory. Participants: Nineteen healthy male college baseball pitchers. Main Outcome Measures: Internal and external hip passive range of motion, hip abduction strength, YBT-LQ composite scores, and kinematic variables of the pitching motion. Results: Stride length demonstrated a moderate positive correlation with dominant limb YBT-LQ composite score (r = .524, P = .02) and nondominant limb YBT-LQ composite score (r = .550, P = .01), and a weak positive correlation with normalized time to maximal humerus velocity (r = .458, P = .04). Stride length had a moderate negative correlation with normalized time to maximal thorax velocity (r = -.522, P = .02) and dominant hip total rotational motion (TRM; r = -.660, P = .002), and had a strong negative correlation with normalized time from stride foot contact to maximal knee flexion (r = -.722, P < .001). Dominant limb YBT-LQ composite score had a weak negative correlation with hip abduction strength difference (r = -.459, P = .04) and normalized time to maximal thorax velocity (r = -.468, P = .04). Nondominant limb YBT-LQ composite score demonstrated a weak negative correlation with normalized time to maximal thorax velocity (r = -.450, P = .05) and had a moderate negative correlation with dominant hip TRM (r = -.668, P = .001). There were no other significant relationships between the remaining variables. Conclusions: YBT-LQ is a clinical measure that can be used to correlate with hip musculoskeletal characteristics and pitching kinematics in NCAA Division I pitchers.


Subject(s)
Baseball/physiology , Hip Joint/physiology , Muscle Strength/physiology , Postural Balance/physiology , Range of Motion, Articular/physiology , Adolescent , Biomechanical Phenomena , Cross-Sectional Studies , Humans , Male , Muscle Strength Dynamometer , Universities , Young Adult
3.
J Diabetes Complications ; 33(1): 23-32, 2019 01.
Article in English | MEDLINE | ID: mdl-30424931

ABSTRACT

Previous studies demonstrated that global deficiency of eNOS in diabetic mice exacerbated renal lesions and that overexpression of eNOS may protect against tissue injury. Our study revealed for the first time overexpression of eNOS leads to disease progression rather than protection. Transgenic mice selectively expressing eNOS in endothelial cells (eNOSTg) were cross bred with Ins2Akita type-1 (AK) diabetic mice to generate eNOS overexpressing eNOSTg/AK mice. Wild type, eNOSTg, AK and eNOSTg/AK mice were assessed for kidney function and blood glucose levels. Remarkably, overexpressing eNOSTg mice showed evidence of unpredicted glomerular injury with segmental mesangiolysis and occasional microaneurysms. Notably, in eNOSTg/AK mice overexpression of eNOS led to increased glomerular/endothelial injury that was associated with increased superoxide levels and renal dysfunction. Results indicate for the first time that overexpressing eNOS in endothelial cells cannot ameliorate diabetic lesions, but paradoxically leads to progression of nephropathy likely due to eNOS uncoupling and superoxide upsurge. This novel finding has a significant impact on current therapeutic strategies to improve endothelial function and prevent progression of diabetic renal disease. Further, the eNOSTg/AK model developed in this study has significant translational potentials for elucidating the underlying mechanism implicated in the deflected function of eNOS in diabetic nephropathy.


Subject(s)
Diabetic Nephropathies/metabolism , Endothelium, Vascular/metabolism , Kidney Glomerulus/metabolism , Nitric Oxide Synthase Type III/biosynthesis , Animals , Diabetic Nephropathies/diagnostic imaging , Diabetic Nephropathies/pathology , Disease Models, Animal , Disease Progression , Endothelium, Vascular/diagnostic imaging , Endothelium, Vascular/pathology , Insulin/genetics , Kidney Glomerulus/diagnostic imaging , Kidney Glomerulus/pathology , Mice , Mice, Inbred Strains , Mice, Transgenic , Microscopy, Electron
4.
Immunity ; 49(2): 275-287.e5, 2018 08 21.
Article in English | MEDLINE | ID: mdl-30054206

ABSTRACT

Airway epithelial cells (AECs) secrete innate immune cytokines that regulate adaptive immune effector cells. In allergen-sensitized humans and mice, the airway and alveolar microenvironment is enriched with colony stimulating factor-1 (CSF1) in response to allergen exposure. In this study we found that AEC-derived CSF1 had a critical role in the production of allergen reactive-IgE production. Furthermore, spatiotemporally secreted CSF1 regulated the recruitment of alveolar dendritic cells (DCs) and enhanced the migration of conventional DC2s (cDC2s) to the draining lymph node in an interferon regulatory factor 4 (IRF4)-dependent manner. CSF1 selectively upregulated the expression of the chemokine receptor CCR7 on the CSF1R+ cDC2, but not the cDC1, population in response to allergen stimuli. Our data describe the functional specification of CSF1-dependent DC subsets that link the innate and adaptive immune responses in T helper 2 (Th2) cell-mediated allergic lung inflammation.


Subject(s)
Allergens/immunology , Dendritic Cells/immunology , Macrophage Colony-Stimulating Factor/immunology , Receptors, CCR7/biosynthesis , Respiratory Mucosa/cytology , Respiratory Mucosa/immunology , Animals , Cell Line , Cell Movement/immunology , Dendritic Cells/classification , Epithelial Cells/cytology , Epithelial Cells/immunology , Humans , Immunity, Innate/immunology , Immunoglobulin E/immunology , Interferon Regulatory Factors/immunology , Lymph Nodes/cytology , Mice , Mice, Inbred C57BL , Mice, Transgenic , RAW 264.7 Cells , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/antagonists & inhibitors , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Th2 Cells/immunology , Up-Regulation/immunology
5.
Am J Transplant ; 18(5): 1247-1255, 2018 05.
Article in English | MEDLINE | ID: mdl-29314558

ABSTRACT

The colony-stimulating factor 1 (CSF1) regulates the differentiation and function of tissue macrophages and determines the outcome of the immune response. The molecular mechanisms behind CSF1-mediated macrophage development remain to be elucidated. Here we demonstrate that neutrophil-derived CSF1 controls macrophage polarization and proliferation, which is necessary for the induction of tolerance. Inhibiting neutrophil production of CSF1 or preventing macrophage proliferation, using targeted nanoparticles loaded with the cell cycle inhibitor simvastatin, abrogates the induction of tolerance. These results provide new mechanistic insights into the developmental requirements of tolerogenic macrophages and identify CSF1 producing neutrophils as critical regulators of the immunological response.


Subject(s)
Heart Transplantation , Immune Tolerance/immunology , Macrophage Colony-Stimulating Factor/metabolism , Macrophages/immunology , Neutrophils/immunology , Transplantation Tolerance/immunology , Animals , Cell Differentiation , Cell Proliferation , Female , Humans , Macrophages/cytology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Neutrophils/metabolism , Signal Transduction
6.
Case Rep Nephrol ; 2017: 1867349, 2017.
Article in English | MEDLINE | ID: mdl-28487790

ABSTRACT

We present an unusual case of shunt nephritis in a 39-year-old male who presented 21 years after placement of a ventriculoperitoneal (VP) shunt. He complained of fevers, headaches, dizziness, and urticarial plaques on arms, trunks, and legs and was found to have anemia, low complement levels, elevated serum creatinine, proteinuria, and new onset microhematuria. Blood and urine cultures were negative. Renal biopsy showed features of acute tubulointerstitial nephritis attributed to vancomycin use. Glomeruli showed increased mesangial hypercellularity and segmental endocapillary proliferation. Immunofluorescence showed focal IgM and C3 staining. Electron microscopy revealed small subendothelial electron-dense deposits. Symptoms and renal insufficiency appeared to improve with antibiotic therapy. He was discharged and readmitted 2 months later with similar presentation. CSF grew Propionibacterium acnes and shunt hardware grew coagulase-negative Staphylococcus. He completed an intravenous antibiotic course and was discharged. On 1-month follow-up, skin lesions persisted but he was otherwise asymptomatic. Follow-up labs showed significant improvement. We did a brief systematic review of the literature on shunt nephritis and report our findings on 79 individual cases. In this review, we comment on the presentation, lab findings, pathological features, and management of this rare, potentially fatal, but curable disease entity.

8.
Nephrol Dial Transplant ; 32(6): 960-968, 2017 06 01.
Article in English | MEDLINE | ID: mdl-27836924

ABSTRACT

Background: Circulating levels of fibroblast growth factor 23 (FGF23) increase progressively and correlate with systemic inflammation in chronic kidney disease (CKD). The aim of this study was to identify and characterize the causal relationship between FGF23 and inflammation in CKD. Methods: Circulating FGF23 and inflammatory cytokines were correlated in healthy subjects and patients with varying levels of CKD. In addition, FGF23 expression in blood and solid organs was measured in normal mice that were exposed acutely (one time) or chronically (2-week) to low-dose lipopolysaccharide (LPS); chronic exposure being either sustained (subcutaneous pellets), intermittent (daily injections) or combined sustained plus acute (subcutaneous pellets plus acute injection on the day of sacrifice). Blood was analyzed for both terminal (cFGF23) and intact (iFGF23) FGF23 levels. Solid tissues were investigated with immunohistochemistry, enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction. Results: FGF23 levels correlated significantly with neutrophil gelatinase-associated lipocalin ( r = 0.72, P < 0.001), C-reactive protein ( r = 0.38, P < 0.001), tumor necrosis factor-α ( r = 0.32, P = 0.001) and interleukin-6 ( r = 0.48, P < 0.001). Acute LPS administration increased tissue FGF23 mRNA and plasma levels of cFGF23 but not iFGF23. Neither chronic sustained nor chronic pulsatile LPS increased the tissue or circulating levels of FGF23. However, acute on chronic LPS raised tissue FGF23 mRNA and both circulating cFG23 and iFGF23. Interestingly, the spleen was the major source of FGF23. Conclusion: Acute on chronic exposure to LPS stimulates FGF23 production in a normal mouse model of inflammation. We provide the first evidence that the spleen, under these conditions, contributes substantially to elevated circulating FGF23 levels.


Subject(s)
Fibroblast Growth Factors/blood , Kidney Failure, Chronic/blood , Lipopolysaccharides/pharmacology , Spleen/metabolism , Animals , Biomarkers/blood , C-Reactive Protein/metabolism , Case-Control Studies , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/immunology , Female , Fibroblast Growth Factor-23 , Humans , Inflammation/metabolism , Interleukin-6/blood , Kidney Failure, Chronic/immunology , Lipocalin-2/blood , Male , Mice , NF-kappa B/metabolism
9.
Oncotarget ; 7(38): 62240-62254, 2016 Sep 20.
Article in English | MEDLINE | ID: mdl-27557496

ABSTRACT

Androgen receptor (AR) and PI3K/AKT/mTORC1 are major survival signals that drive prostate cancer to a lethal disease. Reciprocal activation of these oncogenic pathways from negative cross talks contributes to low/limited success of pathway-selective inhibitors in curbing prostate cancer progression. We report that the antibiotic salinomycin, a cancer stem cell blocker, is a dual-acting AR and mTORC1 inhibitor, inhibiting PTEN-deficient castration-sensitive and castration-resistant prostate cancer in culture and xenograft tumors. AR expression, its transcriptional activity, and androgen biosynthesis regulating enzymes CYP17A1, HSD3ß1 were reduced by sub-micro molar salinomycin. Estrogen receptor-α expression was unchanged. Loss of phosphorylated AR at serine-81, which is an index for nuclear AR activity, preceded total AR reduction. Rapamycin enhanced the AR protein level without altering phosphoAR-Ser81 and CYP17A1. Inactivation of mTORC1, evident from reduced phosphorylation of mTOR and downstream effectors, as well as AMPK activation led to robust autophagy induction. Apoptosis increased modestly, albeit significantly, by sub-micro molar salinomycin. Enhanced stimulatory TSC2 phosphorylation at Ser-1387 by AMPK, and reduced inhibitory TSC2 phosphorylation at Ser-939/Thr-1462 catalyzed by AKT augmented TSC2/TSC1 activity, which led to mTORC1 inhibition. AMPK-mediated raptor phosphorylation further reduced mTOR's kinase function and mTORC1 activity. Our novel finding on dual inhibition of AR and mTORC1 suggests that salinomycin is potentially active as monotherapy against advanced prostate cancer.


Subject(s)
Androgen Receptor Antagonists/pharmacology , Antibiotics, Antineoplastic/pharmacology , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Prostatic Neoplasms/drug therapy , Pyrans/pharmacology , Receptors, Androgen/metabolism , AMP-Activated Protein Kinases/metabolism , Androgen Receptor Antagonists/therapeutic use , Animals , Antibiotics, Antineoplastic/therapeutic use , Apoptosis , Cell Line, Tumor , Cell Proliferation , Estrogen Receptor alpha/metabolism , Humans , Male , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Mice, Nude , Multienzyme Complexes/metabolism , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases , Phosphorylation , Progesterone Reductase/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/genetics , Proto-Oncogene Proteins c-akt/metabolism , Pyrans/therapeutic use , Serine/metabolism , Signal Transduction , Sirolimus/pharmacology , Steroid 17-alpha-Hydroxylase/metabolism , Steroid Isomerases/metabolism , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins/metabolism , Xenograft Model Antitumor Assays
10.
Clin Nephrol ; 85(1): 57-62, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26521888

ABSTRACT

Tumor induced osteomalacia (TIO) is a rare paraneoplastic syndrome characterized by renal phosphate wasting, hypophosphatemia, and osteomalacia. Fibroblast growth factor (FGF)-23, a phosphatonin i.e., phosphaturia-promoting hormone, is commonly implicated in the pathogenesis of TIO. However, very limited information is available about the circulating levels and clinical significance of other phosphatonins that are expressed by TIO-associated tumors. In addition, identification of the primary tumor constitutes a frequent major challenge in the management of TIO. Here, we report a patient with the clinical diagnosis of TIO with elevated blood levels of the phosphatonins FGF-23 and FGF-7; and extensive but unrewarding radiological search for the primary tumor. In selective venous sampling, both FGF-23 and FGF-7 displayed highest concentrations in the left femoral and iliac veins; although lateralization was much more pronounced for FGF-7 than FGF-23. This laboratory finding allowed us to focus on the left lower extremity as the likely location of the primary tumor. Our case is the first to show that FGF-7 can be analyzed in the circulation and used to assist in the diagnosis and localization of TIO-associated tumors.


Subject(s)
Fibroblast Growth Factor 7/blood , Fibroblast Growth Factors/blood , Neoplasms, Connective Tissue/blood , Paraneoplastic Syndromes/blood , Fibroblast Growth Factor-23 , Humans , Lower Extremity , Male , Middle Aged , Neoplasms, Connective Tissue/diagnosis , Osteomalacia , Paraneoplastic Syndromes/diagnosis
11.
Nat Neurosci ; 19(1): 94-101, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26642091

ABSTRACT

Although microglia have been implicated in nerve injury-induced neuropathic pain, the manner by which injured sensory neurons engage microglia remains unclear. We found that peripheral nerve injury induced de novo expression of colony-stimulating factor 1 (CSF1) in injured sensory neurons. CSF1 was transported to the spinal cord, where it targeted the microglial CSF1 receptor (CSF1R). Cre-mediated sensory neuron deletion of Csf1 completely prevented nerve injury-induced mechanical hypersensitivity and reduced microglial activation and proliferation. In contrast, intrathecal injection of CSF1 induced mechanical hypersensitivity and microglial proliferation. Nerve injury also upregulated CSF1 in motoneurons, where it was required for ventral horn microglial activation and proliferation. Downstream of CSF1R, we found that the microglial membrane adaptor protein DAP12 was required for both nerve injury- and intrathecal CSF1-induced upregulation of pain-related microglial genes and the ensuing pain, but not for microglial proliferation. Thus, both CSF1 and DAP12 are potential targets for the pharmacotherapy of neuropathic pain.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Proliferation , Gene Expression Regulation , Macrophage Colony-Stimulating Factor/metabolism , Microglia/metabolism , Motor Neurons/metabolism , Neuralgia/metabolism , Peripheral Nerve Injuries/metabolism , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Sensory Receptor Cells/metabolism , Animals , Male , Mice , Up-Regulation
12.
Kidney Int ; 88(6): 1274-1282, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26422503

ABSTRACT

Infiltrating cells play an important role in both the development of and recovery from acute kidney injury (AKI). Macrophages and renal dendritic cells are of particular interest because they can exhibit distinctly different functional phenotypes, broadly characterized as proinflammatory (M1) or tissue reparative (M2). Resident renal macrophages and dendritic cells participate in recovery from AKI in response to either ischemia/reperfusion or a model of selective proximal tubule injury induced by diphtheria-toxin-induced apoptosis in transgenic mice expressing the human diphtheria toxin receptor on proximal tubule cells. Colony-stimulating factor-1 (CSF-1) is an important factor mediating the recovery from AKI, and CSF-1 can stimulate macrophage and dendritic cell proliferation and polarization during the recovery phase of AKI. The kidney, and specifically the proximal tubule, is a major source of intrarenal CSF-1 production in response to AKI. We induced selective deletion of proximal tubule CSF-1 to determine its role in expansion and proliferation of renal macrophages and dendritic cells and in recovery from AKI. In both models of AKI, there was decreased M2 polarization, delayed functional and structural recovery, and increased tubulointerstitial fibrosis. Thus, intrarenal CSF-1 is an important mediator of macrophage/dendritic cell polarization and recovery from AKI.

13.
Prostate ; 75(15): 1790-801, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26332453

ABSTRACT

BACKGROUND: Altered DNA methylation in CpG islands of gene promoters has been implicated in prostate cancer (PCa) progression and can be used to predict disease outcome. In this study, we determine whether methylation changes of androgen biosynthesis pathway (ABP)-related genes in patients' plasma cell-free DNA (cfDNA) can serve as prognostic markers for biochemical recurrence (BCR). METHODS: Methyl-binding domain capture sequencing (MBDCap-seq) was used to identify differentially methylated regions (DMRs) in primary tumors of patients who subsequently developed BCR or not, respectively. Methylation pyrosequencing of candidate loci was validated in cfDNA samples of 86 PCa patients taken at and/or post-radical prostatectomy (RP) using univariate and multivariate prediction analyses. RESULTS: Putative DMRs in 13 of 30 ABP-related genes were found between tumors of BCR (n = 12) versus no evidence of disease (NED) (n = 15). In silico analysis of The Cancer Genome Atlas data confirmed increased DNA methylation of two loci-SRD5A2 and CYP11A1, which also correlated with their decreased expression, in tumors with subsequent BCR development. Their aberrant cfDNA methylation was also associated with detectable levels of PSA taken after patients' post-RP. Multivariate analysis of the change in cfDNA methylation at all of CpG sites measured along with patient's treatment history predicted if a patient will develop BCR with 77.5% overall accuracy. CONCLUSIONS: Overall, increased DNA methylation of SRD5A2 and CYP11A1 related to androgen biosynthesis functions may play a role in BCR after patients' RP. The correlation between aberrant cfDNA methylation and detectable PSA in post-RP further suggests their utility as predictive markers for PCa recurrence. .


Subject(s)
3-Oxo-5-alpha-Steroid 4-Dehydrogenase/genetics , Cholesterol Side-Chain Cleavage Enzyme/genetics , DNA Methylation , Membrane Proteins/genetics , Neoplasm Recurrence, Local/genetics , Prostatic Neoplasms/genetics , Aged , Biomarkers, Tumor/genetics , CpG Islands , Disease-Free Survival , Humans , Male , Middle Aged , Neoplasm Recurrence, Local/pathology , Promoter Regions, Genetic , Prostate/pathology , Prostate/surgery , Prostatectomy , Prostatic Neoplasms/pathology , Prostatic Neoplasms/surgery , Risk Factors
14.
Acta Diabetol ; 52(5): 897-903, 2015 Oct.
Article in English | MEDLINE | ID: mdl-25753172

ABSTRACT

AIMS: To study the potential pathogenic significance of the coexistence of membranous nephropathy, cerebellar degeneration and anti-glutamic acid decarboxylase (GAD) autoantibodies in patients with diabetes. METHODS: We performed a direct immunocytochemistry on human kidney slides, electron microscopy on human kidney biopsy, direct immunofluorescence on human kidney biopsy. Baboon and rat kidney cell lines were fractionated and subjected to western blotting with antibodies to GAD. RESULTS: In this patient we demonstrate the presence of autoantibodies to GAD, which is highly enriched in podocytes plasma membrane and tubular cells of the kidney as well as sub-endothelial IgG and complement C3 deposits in the glomerular basement membrane (GBM). CONCLUSIONS: We hypothesize the existence in this patient of a common autoimmune pathogenic mechanism with GAD as the autoantigenic determinant, underlying cerebellar degeneration and membranous nephropathy.


Subject(s)
Autoantibodies/analysis , Cerebellar Diseases/immunology , Diabetes Mellitus, Type 2/immunology , Diabetic Nephropathies/immunology , Glomerulonephritis, Membranous/immunology , Glutamate Decarboxylase/immunology , Aged , Animals , Cerebellar Ataxia/immunology , Diabetes Mellitus, Type 2/complications , Fluorescent Antibody Technique, Direct , Humans , Immunohistochemistry , Kidney/immunology , Male , Rats
15.
J Bone Miner Res ; 30(3): 436-48, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25270829

ABSTRACT

Connexin (Cx) 43 serves important roles in bone function and development. Targeted deletion of Cx43 in osteoblasts or osteocytes leads to increased osteocyte apoptosis, osteoclast recruitment, and reduced biomechanical properties. Cx43 forms both gap junction channels and hemichannels, which mediate the communication between adjacent cells or between cell and extracellular environments, respectively. Two transgenic mouse models driven by a DMP1 promoter with the overexpression of dominant negative Cx43 mutants were generated to dissect the functional contribution of Cx43 gap junction channels and hemichannels in osteocytes. The R76W mutant blocks the gap junction channel, but not the hemichannel function, and the Δ130-136 mutant inhibits activity of both types of channels. Δ130-136 mice showed a significant increase in bone mineral density compared to wild-type (WT) and R76W mice. Micro-computed tomography (µCT) analyses revealed a significant increase in total tissue and bone area in midshaft cortical bone of Δ130-136 mice. The bone marrow cavity was expanded, whereas the cortical thickness was increased and associated with increased bone formation along the periosteal area. However, there is no significant alteration in the structure of trabecular bone. Histologic sections of the midshaft showed increased apoptotic osteocytes in Δ130-136, but not in WT and R76W, mice which correlated with altered biomechanical and estimated bone material properties. Osteoclasts were increased along the endocortical surface in both transgenic mice with a greater effect in Δ130-136 mice that likely contributed to the increased marrow cavity. Interestingly, the overall expression of serum bone formation and resorption markers were higher in R76W mice. These findings suggest that osteocytic Cx43 channels play distinctive roles in the bone; hemichannels play a dominant role in regulating osteocyte survival, endocortical bone resorption, and periosteal apposition, and gap junction communication is involved in the process of bone remodeling.


Subject(s)
Bone and Bones/anatomy & histology , Cell Survival/physiology , Connexin 43/physiology , Osteocytes/cytology , Animals , Bone Density , Connexin 43/genetics , Mice , Mice, Transgenic
16.
Case Rep Nephrol ; 2014: 868590, 2014.
Article in English | MEDLINE | ID: mdl-25210633

ABSTRACT

We report a case of hydralazine-induced ANCA-associated glomerulonephritis with pulmonary hemorrhage. A 62-year-old Hispanic man with hypertension, who was being treated with hydralazine 100 mg three times a day for four and half years, presented to the hospital with severe anemia. He had acute kidney injury and urinalysis showed proteinuria, dysmorphic RBCs, and rare RBC cast. CT scan of the chest revealed bilateral pulmonary ground-glass infiltrates. Transbronchial biopsy was consistent with pulmonary hemorrhage. Serologic tests showed high titer PR3 ANCA and, to a lesser extent, MPO ANCA. Kidney biopsy revealed focal segmental necrotizing glomerulonephritis with crescents, without evidence of immune complex deposits. Hydralazine was discontinued and the patient was treated with corticosteroids and intravenous cyclophosphamide. At one-year follow-up, he had no symptoms and anemia had resolved. Kidney function improved dramatically. Serology showed undetectable PR3 ANCA and minimally elevated MPO ANCA. To our knowledge, hydralazine-associated PR3 ANCA has not been previously reported. The possibility of ANCA systemic vasculitis should be included in the differential diagnosis of any patient with hydralazine use and pulmonary renal syndrome. This is a potentially life threatening condition requiring prompt cessation of the drug and treatment with glucocorticoids and immunosuppression.

17.
PLoS One ; 9(4): e93696, 2014.
Article in English | MEDLINE | ID: mdl-24691516

ABSTRACT

Osteoporosis is a silent disease, characterized by a porous bone micro-structure that enhances risk for fractures and associated disabilities. Senile, or age-related osteoporosis (SO), affects both men and women, resulting in increased morbidity and mortality. However, cellular and molecular mechanisms underlying senile osteoporosis are not fully known. Recent studies implicate the accumulation of reactive oxygen species (ROS) and increased oxidative stress as key factors in SO. Herein, we show that loss of caspase-2, a cysteine aspartate protease involved in oxidative stress-induced apoptosis, results in total body and femoral bone loss in aged mice (20% decrease in bone mineral density), and an increase in bone fragility (30% decrease in fracture strength). Importantly, we demonstrate that genetic ablation or selective inhibition of caspase-2 using zVDVAD-fmk results in increased numbers of bone-resorbing osteoclasts and enhanced tartrate-resistant acid phosphatase (TRAP) activity. Conversely, transfection of osteoclast precursors with wild type caspase-2 but not an enzymatic mutant, results in a decrease in TRAP activity. We demonstrate that caspase-2 expression is induced in osteoclasts treated with oxidants such as hydrogen peroxide and that loss of caspase-2 enhances resistance to oxidants, as measured by TRAP activity, and decreases oxidative stress-induced apoptosis of osteoclasts. Moreover, oxidative stress, quantified by assessment of the lipid peroxidation marker, 4-HNE, is increased in Casp2-/- bone, perhaps due to a decrease in antioxidant enzymes such as SOD2. Taken together, our data point to a critical and novel role for caspase-2 in maintaining bone homeostasis by modulating ROS levels and osteoclast apoptosis during conditions of enhanced oxidative stress that occur during aging.


Subject(s)
Apoptosis/genetics , Bone and Bones/metabolism , Caspase 2/metabolism , Osteoclasts/metabolism , Osteoporosis/metabolism , Acid Phosphatase , Aldehydes/administration & dosage , Animals , Bone and Bones/pathology , Caspase 2/genetics , Homeostasis/genetics , Isoenzymes , Lipid Peroxidation/genetics , Mice , Osteoclasts/pathology , Osteoporosis/pathology , Oxidative Stress/genetics , Reactive Oxygen Species , Superoxide Dismutase/metabolism , Tartrate-Resistant Acid Phosphatase
18.
Lab Invest ; 94(4): 371-81, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24468794

ABSTRACT

Colony-stimulating factor 1 (CSF1) is essential for osteoclastogenesis that mediates osteolysis in metastatic tumors. Patients with lung cancer have increased CSF1 in serum and high levels are associated with poor survival. Adenocarcinomas metastasize rapidly and many patients suffer from bone metastasis. Lung cancer stem-like cells sustain tumor growth and potentiate metastasis. The purpose of this study was to determine the role of CSF1 in lung cancer bone metastasis and whether inhibition of CSF1 ameliorates the disease. Human lung adenocarcinoma A549 cells were examined in vitro for CSF1/CSF1R. A549-luc cells were injected intracardiac in NOD/SCID mice and metastasis was assessed. To determine the effect of CSF1 knockdown (KD) in A549 cells on bone metastasis, cells were stably transfected with a retroviral vector containing short-hairpin CSF1 (KD) or empty vector (CT). Results showed that A549 cells express CSF1/CSF1R; CSF1 increased their proliferation and invasion, whereas soluble CSF1R inhibited invasion. Mice injected with A549-luc cells showed osteolytic bone lesions 3.5 weeks after injection and lesions increased over 5 weeks. Tumors recapitulated adenocarcinoma morphology and showed osteoclasts along the tumor/bone interface, trabecular, and cortical bone loss. Analyses of KD cells showed decreased CSF1 protein levels, reduced colony formation in soft agar assay, and decreased fraction of stem-like cells. In CSF1KD mice, the incidence of tumor metastasis was similar to controls, although fewer CSF1KD mice had metastasis in both hind limbs. KD tumors showed reduced CSF1 expression, Ki-67+ cells, and osteoclasts. Importantly, there was a low incidence of large tumors >0.1 mm(2) in CSF1KD mice compared with control mice (10% vs 62.5%). This study established a lung osteolytic bone metastasis model that resembles human disease and suggests that CSF1 is a key determinant of cancer stem cell survival and tumor growth. Results may lead to novel strategies to inhibit CSF1 in lung cancer and improve management of bone metastasis.


Subject(s)
Adenocarcinoma/secondary , Bone Neoplasms/secondary , Bone and Bones/pathology , Lung Neoplasms/pathology , Macrophage Colony-Stimulating Factor/metabolism , Adenocarcinoma/metabolism , Animals , Bone Neoplasms/metabolism , Cell Line, Tumor , Gene Knockdown Techniques , Humans , Lung Neoplasms/metabolism , Macrophage Colony-Stimulating Factor/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Metastasis , Neoplasms, Experimental , Neoplastic Stem Cells/physiology , Osteoclasts/physiology , Receptor, Macrophage Colony-Stimulating Factor/metabolism
19.
Mol Endocrinol ; 27(6): 925-39, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23579488

ABSTRACT

An elevated tumor tissue androgen level, which reactivates androgen receptor in recurrent prostate cancer, arises from the intratumor synthesis of 5α-dihydrotestosterone through use of the precursor steroid dehydroepiandrosterone (DHEA) and is fueled by the steroidogenic enzymes 3ß-hydroxysteroid dehydrogenase (3ß-HSD1), aldoketoreductase (AKR1C3), and steroid 5-alpha reductase, type 1 (SRD5A1) present in cancer tissue. Sulfotransferase 2B1b (SULT2B1b) (in short, SULT2B) is a prostate-expressed hydroxysteroid SULT that converts cholesterol, oxysterols, and DHEA to 3ß-sulfates. DHEA metabolism involving sulfonation by SULT2B can potentially interfere with intraprostate androgen synthesis due to reduction of free DHEA pool and, thus, conversion of DHEA to androstenedione. Here we report that in prostatectomy specimens from treatment-naive patients, SULT2B expression is markedly reduced in malignant tissue (P < .001, Mann-Whitney U test) compared with robust expression in adjacent nonmalignant glands. SULT2B was detected in formalin-fixed specimens by immunohistochemistry on individual sections and tissue array. Immunoblotting of protein lysates of frozen cancer and matched benign tissue confirmed immunohistochemistry results. An in-house-developed rabbit polyclonal antibody against full-length human SULT2B was validated for specificity and used in the analyses. Ligand-activated vitamin D receptor induced the SULT2B1 promoter in vivo in mouse prostate and increased SULT2B mRNA and protein levels in vitro in prostate cancer cells. A vitamin D receptor/retinoid X receptor-α-bound DNA element (with a DR7 motif) mediated induction of the transfected SULT2B1 promoter in calcitriol-treated cells. SULT2B knockdown caused an increased proliferation rate of prostate cancer cells upon stimulation by DHEA. These results suggest that the tumor tissue SULT2B level may partly control prostate cancer growth, and its induction in a therapeutic setting may inhibit disease progression.


Subject(s)
Prostatic Neoplasms/enzymology , Receptors, Calcitriol/physiology , Sulfotransferases/genetics , Animals , Antibodies/chemistry , Antibodies/immunology , Antibody Specificity , Base Sequence , Calcitriol/physiology , Cell Line, Tumor , Cell Proliferation , DNA Footprinting , Enzyme Induction , Gene Expression , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Mice, Inbred C57BL , Prostate/enzymology , Prostate/pathology , Protein Binding , Response Elements , Sequence Analysis, DNA , Sulfotransferases/immunology , Sulfotransferases/metabolism , Tissue Array Analysis
20.
J Bone Miner Res ; 28(7): 1611-21, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23456878

ABSTRACT

The increased osteocyte death by oxidative stress (OS) during aging is a major cause contributing to the impairment of bone quality and bone loss. However, the underlying molecular mechanism is largely unknown. Here, we show that H2O2 induced cell death of primary osteocytes and osteocytic MLO-Y4 cells, and also caused dose-dependent decreased expression of gap junction and hemichannel-forming connexin 43 (Cx43). The decrease of Cx43 expression was also demonstrated with the treatment of other oxidants, rotenone and menadione. Antioxidant reversed the effects of oxidants on Cx43 expression and osteocyte cell death. Cx43 protein was also much lower in the osteocytes from 20-month-old as opposed to the 5-week-old or 20-week old mice. Dye transfer assay showed that H2O2 reduced the gap junction intercellular communication (GJIC). In contrast to the effect on GJIC, there was a dose-dependent increase of hemichannel function by H2O2, which was correlated with the increased cell surface expression of Cx43. Cx43(E2) antibody, an antibody that specifically blocks Cx43 hemichannel activity but not gap junctions, completely blocked dye uptake induced by H2O2 and further exacerbated H2O2-induced osteocytic cell death. In addition, knockdown of Cx43 expression by small interfering RNA (siRNA) increased the susceptibility of the cells to OS-induced death. Together, our study provides a novel cell protective mechanism mediated by osteocytic Cx43 channels against OS.


Subject(s)
Connexin 43/biosynthesis , Gene Expression Regulation/drug effects , Hydrogen Peroxide/pharmacology , Osteocytes/metabolism , Oxidants/pharmacology , Oxidative Stress/drug effects , Animals , Cell Communication/drug effects , Cell Communication/genetics , Cell Death/drug effects , Cell Line , Connexin 43/genetics , Gap Junctions/genetics , Gap Junctions/metabolism , Gene Expression Regulation/genetics , Mice , Osteocytes/cytology , Oxidative Stress/genetics
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