Publications by authors named "Si-Tse Jiang"

32 Publications

Negative Regulation of the Differentiation of Flk2 CD34 LSK Hematopoietic Stem Cells by EKLF/KLF1.

Int J Mol Sci 2020 Nov 10;21(22). Epub 2020 Nov 10.

Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 115, Taiwan.

Erythroid Krüppel-like factor (EKLF/KLF1) was identified initially as a critical erythroid-specific transcription factor and was later found to be also expressed in other types of hematopoietic cells, including megakaryocytes and several progenitors. In this study, we have examined the regulatory effects of EKLF on hematopoiesis by comparative analysis of E14.5 fetal livers from wild-type and gene knockout (KO) mouse embryos. Depletion of EKLF expression greatly changes the populations of different types of hematopoietic cells, including, unexpectedly, the long-term hematopoietic stem cells Flk2 CD34 Lin Sca1 c-Kit (LSK)-HSC. In an interesting correlation, is expressed at a relatively high level in multipotent progenitor (MPP). Furthermore, EKLF appears to repress the expression of the colony-stimulating factor 2 receptor β subunit (CSF2RB). As a result, Flk2 CD34 LSK-HSC gains increased differentiation capability upon depletion of EKLF, as demonstrated by the methylcellulose colony formation assay and by serial transplantation experiments in vivo. Together, these data demonstrate the regulation of hematopoiesis in vertebrates by EKLF through its negative regulatory effects on the differentiation of the hematopoietic stem and progenitor cells, including Flk2 CD34 LSK-HSCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21228448DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697791PMC
November 2020

Kidney-based in vivo model for drug-induced nephrotoxicity testing.

Sci Rep 2020 08 14;10(1):13640. Epub 2020 Aug 14.

Division of Pediatric Nephrology, Department of Pediatrics, National Cheng Kung University and Hospital, 138 Sheng-Li Road, Tainan, 70403, Taiwan.

The need is critical and urgent for a real-time, highly specific, and sensitive acute kidney injury biomarker. This study sought to establish a sensitive and specific Miox-NanoLuc transgenic mouse for early detection of drug-induced nephrotoxicity. We generated Miox-NanoLuc transgenic mice with kidney-specific NanoLuc overexpression. Our data showed that Miox-NanoLuc-produced luminescence was kidney-specific and had good stability at room temperature, 4 °C, - 20 °C, and repeated freeze-thaw cycles. Serum levels of BUN and creatinine were significantly increased at day 2 or 3 in cisplatin-treated mice and at day 5 in aristolochic acid (AAI)-treated mice. Particularly, the serum and urine Miox-NanoLuc luminescence levels were significantly increased at day 1 in cisplatin-treated mice and at day 3 in AAI-treated mice. Renal pathological analysis showed that the kidney sections of cisplatin-treated mice at day 5 and AAI-treated mice at day 13 showed cytolysis and marked vacuolization of tubular cells. In conclusion, we developed a new platform to early quantify drug-induced nephrotoxicity before serum BUN and creatinine levels increased and pathological tubular cell injury occurred. This model may serve as an early detection for drug- and food-induced nephrotoxicity and as an animal model to investigate tubular cell injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-70502-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7428004PMC
August 2020

A reporter mouse for non-invasive detection of toll-like receptor ligands induced acute phase responses.

Sci Rep 2019 12 13;9(1):19065. Epub 2019 Dec 13.

National Laboratory Animal Center, National Applied Research Laboratories, Taipei, Taiwan.

The acute phase response (APR) is a systemic first-line defense against challenges including infection, trauma, stress, and neoplasia. Alteration of acute phase protein (APP) levels in plasma is the most important change during acute phase response. C-reactive protein (CRP), which increases dramatically during inflammation onset, is an indicator of inflammation. To monitor the process of APR, we generated human CRP promoter-driven luciferase transgenic (hCRP-Luc) mice to quantify the hCRP promoter activation in vivo. The naïve female hCRP-Luc mice express low basal levels of liver bioluminescence, but the naïve male hCRP-Luc mice do not. Thus, female hCRP-Luc mice are suitable for monitoring the process of APR. The liver bioluminescence of female hCRP-Luc mice can be induced by several toll-like receptor (TLR) ligands. The expression of liver bioluminescence was highly sensitive to endotoxin stimulation in a dose-dependent manner. On-off-on bioluminescence response was noted in female hCRP-Luc mice upon two endotoxin stimulations one month apart. The LPS-induced bioluminescence of the female hCRP-Luc mice was IL-6-mediated and associated with APP alpha-1-acid glycoprotein expression. In conclusion, the female hCRP-Luc mouse is a non-invasive, sensitive and reusable reporter tool for APR.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-55281-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910947PMC
December 2019

Prothymosin α promotes STAT3 acetylation to induce cystogenesis in Pkd1-deficient mice.

FASEB J 2019 11 5;33(11):13051-13061. Epub 2019 Oct 5.

Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

Polycystic kidney disease (PKD) is characterized by the expansion of fluid-filled cysts in the kidney, which impair the function of kidney and eventually leads to end-stage renal failure. It has been previously demonstrated that transgenic overexpression of prothymosin α (ProT) induces the development of PKD; however, the underlying mechanisms remain unclear. In this study, we used a mouse PKD model that sustains kidney-specific low-expression of to illustrate that aberrant up-regulation of ProT occurs in cyst-lining epithelial cells, and we further developed an cystogenesis model to demonstrate that the suppression of ProT is sufficient to reduce cyst formation. Next, we found that the expression of ProT was accompanied with prominent augmentation of protein acetylation in PKD, which results in the activation of downstream signal transducer and activator of transcription (STAT) 3. The pathologic role of STAT3 in PKD has been previously reported. We determined that this molecular mechanism of protein acetylation is involved with the interaction between ProT and STAT3; consequently, it causes the deprivation of histone deacetylase 3 from the indicated protein. Conclusively, these results elucidate the significant role of ProT, including protein acetylation and STAT3 activation in PKD, which represent potential for ameliorating the disease progression of PKD.-Chen, Y.-C., Su, Y.-C., Shieh, G.-S., Su, B.-H., Su, W.-C., Huang, P.-H., Jiang, S.-T., Shiau, A.-L., Wu, C.-L. Prothymosin α promotes STAT3 acetylation to induce cystogenesis in Pkd1-deficient mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201900504RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6902686PMC
November 2019

Targeted Delivery of Curcumin Rescues Endoplasmic Reticulum-Retained Mutant NOX2 Protein and Avoids Leukocyte Apoptosis.

J Immunol 2019 06 13;202(12):3394-3403. Epub 2019 May 13.

Institute of Clinical Medicine, National Cheng Kung University, Tainan 70457, Taiwan;

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by defects in the leukocyte NADP oxidase. We previously reported that sarcoplasmic/endoplasmic reticulum calcium pump (SERCA) inhibitors could be used to rescue mutant H338Y-gp91phox protein of a particular type of CGD with a mutation, leading to endoplasmic reticulum (ER) retention of the mutant protein. In this study, we developed a novel mouse model with the mutation on a Cybb knockout background and investigated the therapeutic effects of ER-targeted delivery of the SERCA inhibitor, curcumin, with poly(lactic-coglycolic acid) (PLGA) nanoparticles (NPs). We found that PLGA encapsulation improved the efficacy of curcumin as a SERCA inhibitor to induce ER calcium release. ER-targeting curcumin-loaded PLGA NPs reduced and delayed extracellular calcium entry and protected the cells from mitochondrial damage and apoptosis. In vivo studies showed that ER-targeting curcumin-loaded PLGA NPs treatment enhanced neutrophil gp91phox expression, ROS production and peritoneal bacterial clearance ability of the transgenic mice. Our findings indicate that ER-targeted delivery of curcumin not only rescues ER-retained H338Y-gp91phox protein, and hence leukocyte function, but also enhances the bioavailability and reduces cytotoxicity. Modulation of ER function by using organelle-targeted NPs may be a promising strategy to improve the therapeutic potential of curcumin as a treatment for CGD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1801599DOI Listing
June 2019

MST3 is involved in ENaC-mediated hypertension.

Am J Physiol Renal Physiol 2019 07 10;317(7):F30-F42. Epub 2019 Apr 10.

School of Pharmacy, China Medical University , Taichung , Taiwan.

Liddle syndrome is an inherited form of human hypertension caused by increasing epithelial Na channel (ENaC) expression. Increased Na retention through ENaC with subsequent volume expansion causes hypertension. In addition to ENaC, the Na-K-Cl cotransporter (NKCC) and Na-Cl symporter (NCC) are responsible for Na reabsorption in the kidneys. Several Na transporters are evolutionarily regulated by the Ste20 kinase family. Ste20-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 phosphorylate downstream NKCC2 and NCC to maintain Na and blood pressure (BP) homeostasis. Mammalian Ste20 kinase 3 (MST3) is another member of the Ste20 family. We previously reported that reduced MST3 levels were found in the kidneys in spontaneously hypertensive rats and that MST3 was involved in Na regulation. To determine whether MST3 is involved in BP stability through Na regulation, we generated a MST3 hypomorphic mutation and designated MST3 and MST3 mice to examine BP and serum Na and K concentrations. MST3 mice exhibited hypernatremia, hypokalemia, and hypertension. The increased ENaC in the kidney played roles in hypernatremia. The reabsorption of more Na promoted more K secretion in the kidney and caused hypokalemia. The hypernatremia and hypokalemia in MST3 mice were significantly reversed by the ENaC inhibitor amiloride, indicating that MST3 mice reabsorbed more Na through ENaC. Furthermore, Madin-Darby canine kidney cells stably expressing kinase-dead MST3 displayed elevated ENaC currents. Both the in vivo and in vitro results indicated that MST3 maintained Na homeostasis through ENaC regulation. We are the first to report that MST3 maintains BP stability through ENaC regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajprenal.00455.2018DOI Listing
July 2019

A Novel Murine Model Expressing a Chimeric mSCARB2/hSCARB2 Receptor Is Highly Susceptible to Oral Infection with Clinical Isolates of Enterovirus 71.

J Virol 2019 06 15;93(11). Epub 2019 May 15.

Graduate Institute of Biomedical Science, Chang Gung University, Guishan, Taoyuan, Taiwan

Enterovirus 71 (EV71) infection is generally associated with hand-foot-and-mouth disease (HFMD) and may cause severe neurological disorders and even death. An effective murine oral infection model for studying the pathogenesis of various clinical EV71 isolates is lacking. We developed a transgenic (Tg) mouse that expresses an EV71 receptor, that is, human scavenger receptor class B member 2 (hSCARB2), in a pattern highly similar to that of endogenous murine SCARB2 (mSCARB2) protein. A FLAG-tagged cDNA fragment composed of exons 3 to 12 was inserted into a murine gene-containing bacterial artificial chromosome (BAC) clone, and the resulting transgene was used for establishment of chimeric receptor-expressing Tg mice. Tg mice intragastrically (i.g.) infected with clinical isolates of EV71 showed neurological symptoms, such as ataxia and paralysis, and fatality. There was an age-dependent decrease in susceptibility to viral infection. Pathological characteristics of the infected Tg mice resembled those of encephalomyelitis in human patients. Viral infection was accompanied by microglial activation. Clodronate treatment of the brain slices from Tg mice enhanced viral replication, while lipopolysaccharide treatment significantly inhibited it, suggesting an antiviral role for microglia during EV71 infection. Taken together, this Tg mouse provides a model that closely mimics natural infection for studying EV71 pathogenesis and for evaluating the efficacy of vaccines or other antiviral drugs. The availability of a murine model of EV71 infection is beneficial for the understanding of pathogenic mechanisms and the development and assessment of vaccines and antiviral drugs. However, the lack of a murine oral infection model thwarted the study of pathogenesis induced by clinically relevant EV71 strains that are transmitted via the oral-oral or oral-fecal route. Our Tg mice could be intragastrically infected with clinically relevant EV71 strains in an efficient way and developed neurological symptoms and pathological changes strikingly resembling those of human infection. Moreover, these mice showed an age-dependent change in susceptibility that is similar to the human case. This Tg mouse, when combined with the use of other genetically modified mice, potentially contributes to studying the relationship between developmental changes in immunity and susceptibility to virus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00183-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6532076PMC
June 2019

Safe Nanocomposite-Mediated Efficient Delivery of MicroRNA Plasmids for Autosomal Dominant Polycystic Kidney Disease (ADPKD) Therapy.

Adv Healthc Mater 2019 03 23;8(5):e1801358. Epub 2019 Jan 23.

Department of Chemistry, Chinese Culture University, 55, Hwa-Kang Road, Yang-Ming-Shan, Taipei, 11114, Taiwan.

There is currently no cure for gene mutation-caused autosomal dominant polycystic kidney disease (ADPKD). Over half of patients with ADPKD eventually develop kidney failure, requiring dialysis or kidney transplantation. Current treatment modalities for ADPKD focus on reducing morbidity and mortality from renal and extrarenal complications of the disease. MicroRNA has been shown to be useful in treating ADPKD. This study combines anti-miRNA plasmids and iron oxide/alginate nanoparticles for conjugation with antikidney antibodies. These nanocomposites can specifically target renal tubular cells, providing a potential treatment for ADPKD. Magnetic resonance imaging and in vivo imaging system results show effective targeting of renal cells. Anti-miRNA plasmids released from the nanocomposites inhibit cell proliferation and cyst formation in the PKD cellular and animal models. The results suggest the novel combination of the anti-miRNA plasmids and nanomaterials provides potential clinical implications for ADPKD treatment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/adhm.201801358DOI Listing
March 2019

CK1α ablation in keratinocytes induces p53-dependent, sunburn-protective skin hyperpigmentation.

Proc Natl Acad Sci U S A 2017 09 6;114(38):E8035-E8044. Epub 2017 Sep 6.

The Lautenberg Center for Immunology and Cancer Research, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel

Casein kinase 1α (CK1α), a component of the β-catenin destruction complex, is a critical regulator of Wnt signaling; its ablation induces both Wnt and p53 activation. To characterize the role of CK1α (encoded by ) in skin physiology, we crossed mice harboring floxed with mice expressing K14-Cre-ER to generate mice in which tamoxifen induces the deletion of exclusively in keratinocytes [single-knockout (SKO) mice]. As expected, CK1α loss was accompanied by β-catenin and p53 stabilization, with the preferential induction of p53 target genes, but phenotypically most striking was hyperpigmentation of the skin, importantly without tumorigenesis, for at least 9 mo after ablation. The number of epidermal melanocytes and eumelanin levels were dramatically increased in SKO mice. To clarify the putative role of p53 in epidermal hyperpigmentation, we established K14-Cre-ER CK1α/p53 double-knockout (DKO) mice and found that coablation failed to induce epidermal hyperpigmentation, demonstrating that it was p53-dependent. Transcriptome analysis of the epidermis revealed p53-dependent up-regulation of Kit ligand (KitL). SKO mice treated with ACK2 (a Kit-neutralizing antibody) or imatinib (a Kit inhibitor) abrogated the CK1α ablation-induced hyperpigmentation, demonstrating that it requires the KitL/Kit pathway. Pro-opiomelanocortin (POMC), a precursor of α-melanocyte-stimulating hormone (α-MSH), was not activated in the CK1α ablation-induced hyperpigmentation, which is in contrast to the mechanism of p53-dependent UV tanning. Nevertheless, acute sunburn effects were successfully prevented in the hyperpigmented skin of SKO mice. CK1α inhibition induces skin-protective eumelanin but no carcinogenic pheomelanin and may therefore constitute an effective strategy for safely increasing eumelanin via UV-independent pathways, protecting against acute sunburn.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1702763114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617257PMC
September 2017

The type VI adenylyl cyclase protects cardiomyocytes from β-adrenergic stress by a PKA/STAT3-dependent pathway.

J Biomed Sci 2017 Sep 4;24(1):68. Epub 2017 Sep 4.

Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.

Background: The type VI adenylyl cyclase (AC6) is a main contributor of cAMP production in the heart. The amino acid (aa) sequence of AC6 is highly homologous to that of another major cardiac adenylyl cyclase, AC5, except for its N-terminus (AC6-N, aa 1-86). Activation of AC6, rather than AC5, produces cardioprotective effects against heart failure, while the underlying mechanism remains to be unveiled. Using an AC6-null (AC6) mouse and a knockin mouse with AC6-N deletion (AC6 ), we aimed to investigate the cardioprotective mechanism of AC6 in the heart.

Methods: Western blot analysis and immunofluorescence staining were performed to determine the intracellular distribution of AC6, AC6-ΔN (a truncated AC6 lacking the first 86 amino acids), and STAT3 activation. Activities of AC6 and AC6-ΔN in the heart were assessed by cAMP assay. Apoptosis of cardiomyocytes were evaluated by the TUNEL assay and a propidium iodine-based survival assay. Fibrosis was examined by collagen staining.

Results: Immunofluorescence staining revealed that cardiac AC6 was mainly anchored on the sarcolemmal membranes, while AC6-ΔN was redistributed to the sarcoplasmic reticulum. AC6 and AC6 mice had more apoptotic myocytes and cardiac remodeling than WT mice in experimental models of isoproterenol (ISO)-induced myocardial injury. Adult cardiomyocytes isolated from AC6 or AC6 mice survived poorly after exposure to ISO, which produced no effect on WT cardiomyocytes under the condition tested. Importantly, ISO treatment induced cardiac STAT3 phosphorylation/activation in WT mice, but not in AC6 and AC6 mice. Pharmacological blockage of PKA-, Src-, or STAT3- pathway markedly reduced the survival of WT myocytes in the presence of ISO, but did not affect those of AC6 and AC6 myocytes, suggesting an important role of AC6 in mediating cardioprotective action through the activation of PKA-Src-STAT3-signaling.

Conclusions: Collectively, AC6-N controls the anchorage of cardiac AC6 on the sarcolemmal membrane, which enables the coupling of AC6 with the pro-survival PKA-STAT3 pathway. Our findings may facilitate the development of novel therapies for heart failure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12929-017-0367-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5584049PMC
September 2017

Overexpression of exogenous kidney-specific Ngal attenuates progressive cyst development and prolongs lifespan in a murine model of polycystic kidney disease.

Kidney Int 2017 02 27;91(2):412-422. Epub 2016 Oct 27.

Department of Life Science, National Taiwan Normal University, Taipei, Taiwan. Electronic address:

Neutrophil gelatinase-associated lipocalin (Ngal) is a biomarker for acute and chronic renal injuries, including polycystic kidney disease (PKD). However, the effect of Ngal on PKD progression remains unexplored. To study this, we generated 3 strains of mice with different expression levels of Ngal within an established PKD model (Pkd1): Pkd1 (with endogenous Ngal), Pkd1; Ngal (with endogenous and overexpression of exogenous kidney-specific Ngal) and Pkd1; Ngal mice (with Ngal deficiency). Knockout of endogenous Ngal had no effect on phenotypes, cystic progression, or survival of the PKD mice. However, the transgenic mice had a significantly longer lifespan, smaller (but not fewer) renal cysts, and less interstitial fibrosis than the mice without or with endogenous Ngal. Western-blot analyses showed significant increases in Ngal and cleaved caspase-3 and decreases in α-smooth muscle actin, hypoxia-inducible factor 1-α, pro-caspase 3, proliferating cell nuclear antigen, Akt, mammalian target of rapamycin, and S6 Kinase in the transgenic mice as compared with the other 2 strains of PKD mice. Thus, overexpression of exogenous kidney-specific Ngal reduced cystic progression and prolonged the lifespan in PKD mice, was associated with reductions in interstitial fibrosis and proliferation, and augmented apoptosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.kint.2016.09.005DOI Listing
February 2017

Deficiency of CPEB2-Confined Choline Acetyltransferase Expression in the Dorsal Motor Nucleus of Vagus Causes Hyperactivated Parasympathetic Signaling-Associated Bronchoconstriction.

J Neurosci 2016 12 3;36(50):12661-12676. Epub 2016 Nov 3.

Institute of Biomedical Sciences, Academia Sinica, 11529 Taipei, Taiwan,

Cytoplasmic polyadenylation element binding protein 2 (CPEB2) is an RNA-binding protein and translational regulator. To understand the physiological function of CPEB2, we generated CPEB2 knock-out (KO) mice and found that most died within 3 d after birth. CPEB2 is highly expressed in the brainstem, which controls vital functions, such as breathing. Whole-body plethysmography revealed that KO neonates had aberrant respiration with frequent apnea. Nevertheless, the morphology and function of the respiratory rhythm generator and diaphragm neuromuscular junctions appeared normal. We found that upregulated translation of choline acetyltransferase in the CPEB2 KO dorsal motor nucleus of vagus resulted in hyperactivation of parasympathetic signaling-induced bronchoconstriction, as evidenced by increased pulmonary acetylcholine and phosphorylated myosin light chain 2 in bronchial smooth muscles. Specific deletion of CPEB2 in cholinergic neurons sufficiently caused increased apnea in neonatal pups and airway hyper-reactivity in adult mice. Moreover, inhalation of an anticholinergic bronchodilator reduced apnea episodes in global and cholinergic CPEB2-KO mice. Together, the elevated airway constriction induced by cholinergic transmission in KO neonates may account for the respiratory defect and mortality.

Significance Statement: This study first generated and characterized cpeb2 gene-deficient mice. CPEB2-knock-out (KO) mice are born alive but most die within 3 d after birth showing no overt defects in anatomy. We found that the KO neonates showed severe apnea and altered respiratory pattern. Such respiratory defects could be recapitulated in mice with pan-neuron-specific or cholinergic neuron-specific ablation of the cpeb2 gene. Further investigation revealed that cholinergic transmission in the KO dorsal motor nucleus of vagus was overactivated because KO mice lack CPEB2-suppressed translation of the rate-limiting enzyme in the production of acetylcholine (i.e., choline acetyltransferase). Consequently, increased parasympathetic signaling leads to hyperactivated bronchoconstriction and abnormal respiration in the KO neonates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.0557-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6705661PMC
December 2016

Early Detection of T cell Transfer-induced Autoimmune Colitis by In Vivo Imaging System.

Sci Rep 2016 10 20;6:35635. Epub 2016 Oct 20.

National Laboratory Animal Center, National Applied Research Laboratories, Taipei, Taiwan.

Inflammatory bowel disease is a chronic and progressive inflammatory intestinal disease that includes two major types, namely ulcerative colitis and Crohn's disease (CD). CD is characterized by intestinal epithelial hyperplasia and inflammatory cell infiltration. Transfer of CD25CD45RBCD4 (naïve) T cells into immunodeficiency mice induces autoimmune colitis with pathological lesions similar to CD and loss of body weight 4 weeks after cell transfer. However, weight loss neither has sufficient sensitivity nor totally matches the pathological findings of CD. To establish an early and sensitive indicator of autoimmune colitis model, the transferred T cell-induced colitis mouse model was modified by transferring luciferase-expressing donor T cells and determining the colitis by in vivo imaging system (IVIS). Colitis was detected with IVIS 7-10 days before the onset of body weight loss and diarrhea. IVIS was also applied in the dexamethasone treatment trial, and was a more sensitive indicator than body weight changes. All IVIS signals were parallel to the pathological abnormalities of the gut and immunological analysis results. In summary, IVIS provides both sensitive and objective means to monitor the disease course of transferred T cell-induced CD and fulfills the 3Rs principle of humane care of laboratory animals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep35635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5071899PMC
October 2016

Cul3-KLHL20 Ubiquitin Ligase Governs the Turnover of ULK1 and VPS34 Complexes to Control Autophagy Termination.

Mol Cell 2016 Jan 10;61(1):84-97. Epub 2015 Dec 10.

Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan; Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei 106, Taiwan; Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan. Electronic address:

Autophagy, a cellular self-eating mechanism, is important for maintaining cell survival and tissue homeostasis in various stressed conditions. Although the molecular mechanism of autophagy induction has been well studied, how cells terminate autophagy process remains elusive. Here, we show that ULK1, a serine/threonine kinase critical for autophagy initiation, is a substrate of the Cul3-KLHL20 ubiquitin ligase. Upon autophagy induction, ULK1 autophosphorylation facilitates its recruitment to KLHL20 for ubiquitination and proteolysis. This autophagy-stimulated, KLHL20-dependent ULK1 degradation restrains the amplitude and duration of autophagy. Additionally, KLHL20 governs the degradation of ATG13, VPS34, Beclin-1, and ATG14 in prolonged starvation through a direct or indirect mechanism. Impairment of KLHL20-mediated regulation of autophagy dynamics potentiates starvation-induced cell death and aggravates diabetes-associated muscle atrophy. Our study identifies a key role of KLHL20 in autophagy termination by controlling autophagy-dependent turnover of ULK1 and VPS34 complex subunits and reveals the pathophysiological functions of this autophagy termination mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2015.11.001DOI Listing
January 2016

Insulin Receptor Substrate-1 Activation Mediated p53 Downregulation Protects Against Hypoxic-Ischemia in the Neonatal Brain.

Mol Neurobiol 2016 08 26;53(6):3658-3669. Epub 2015 Jun 26.

Department of Pediatrics, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

This study determined if dietary restriction (DR) protects against hypoxic-ischemia (HI) in the neonatal brain via insulin receptor substrate-1 (IRS-1)/Akt pathway-mediated downregulation of p53 in the neurovascular unit. On postnatal (P) day 7, HI was induced in rat pups grouped from P1 into normal litter size (NL, 12 pups/dam) and increased litter size (DR, 18 pups/dam). In vivo IRS-1 anti-sense oligonucleotide and IRS-1 overexpressed recombinant adenovirus were given, and neurovascular damage was assessed. In vitro models of oxygen-glucose deprivation (OGD) examined the inhibition and overexpression of IRS-1 on p53 and cell death in neurons and endothelial cells. Compared to NL pups, DR pups had significantly higher IRS-1, p-IRS-1, and pAkt levels, decreased p53, more tight junction proteins, reduced blood-brain barrier (BBB) damage after HI, and less infarct volumes at P21. Immunofluorescence revealed that IRS-1 was upregulated in the endothelial cells and neurons of DR pups. IRS-1 downregulation in DR pups reduced p-Akt, increased p53, worsened BBB damage, and increased brain injury, whereas IRS-1 overexpression in NL pups upregulated p-Akt, decreased p53, attenuated BBB damage, and decreased brain injury. In vitro, IRS-1 downregulation aggravated cell death in neurons and endothelial cells and is associated with decreased p-Akt and increased p53. In contrast, IRS-1 overexpression reduced cell death in endothelial cells with increased p-Akt and decreased p53. In conclusion, DR reduces neurovascular damage after HI in the neonatal brain through an IRS-1/Akt-mediated p53 downregulation, suggesting that IRS-1 signaling is a therapeutic target for hypoxic brain injury in neonates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12035-015-9300-5DOI Listing
August 2016

Deltex1 antagonizes HIF-1α and sustains the stability of regulatory T cells in vivo.

Nat Commun 2015 Feb 19;6:6353. Epub 2015 Feb 19.

1] Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, R.O.C [2] Graduate Institute of Immunology, National Taiwan University, Taipei 10057, Taiwan, R.O.C.

Application of regulatory T cells (Tregs) in transplantation, autoimmunity and allergy has been extensively explored, but how Foxp3 and Treg stability is regulated in vivo is incompletely understood. Here, we identify a requirement for Deltex1 (DTX1), a contributor to T-cell anergy and Foxp3 protein level maintenance in vivo. Dtx1(-/-) Tregs are as effective as WT Tregs in the inhibition of CD4(+)CD25(-) T-cell activation in vitro. However, the suppressive ability of Dtx1(-/-) Tregs is greatly impaired in vivo. We find that Foxp3 expression is diminished when Dtx1(-/-) Tregs are co-transferred with effector T cells in vivo. DTX1 promotes the degradation of HIF-1α. Knockout of HIF-1α restores the Foxp3 stability and rescues the defective suppressive activity in Dtx1(-/-) Treg cells in vivo. Our results suggest that DTX1 exerts another level of control on Treg stability in vivo by sustaining the expression of Foxp3 protein in Tregs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncomms7353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4346631PMC
February 2015

Adipocyte IL-15 regulates local and systemic NK cell development.

J Immunol 2014 Aug 9;193(4):1747-58. Epub 2014 Jul 9.

Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 11490, Taiwan; and Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan

NK cell development and homeostasis require IL-15 produced by both hematopoietic and parenchymal cells. Certain hematopoietic IL-15 sources, such as macrophages and dendritic cells, are known, whereas the source of parenchymal IL-15 remains elusive. Using two types of adipocyte-specific Il15(-/-) mice, we identified adipocytes as a parenchymal IL-15 source that supported NK cell development nonredundantly. Both adipocyte-specific Il15(-/-) mice showed reduced IL-15 production specifically in the adipose tissue but impaired NK cell development in the spleen and liver in addition to the adipose tissue. We also found that the adipose tissue harbored NK progenitors as other niches (e.g. spleen) for NK cell development, and that NK cells derived from transplanted adipose tissue populated the recipient's spleen and liver. These findings suggest that adipocyte IL-15 contributes to systemic NK cell development by supporting NK cell development in the adipose tissue, which serves as a source of NK cells for other organs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1400868DOI Listing
August 2014

Misregulated progesterone secretion and impaired pregnancy in Cyp11a1 transgenic mice.

Biol Reprod 2013 Oct 17;89(4):91. Epub 2013 Oct 17.

Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.

Normal pregnancy is supported by increased levels of progesterone (P4), which is secreted from ovarian luteal cells via enzymatic steps catalyzed by P450scc (CYP11A1) and HSD3B. The development and maintenance of corpora lutea during pregnancy, however, are less well understood. Here we used Cyp11a1 transgenic mice to delineate the steps of luteal cell differentiation during pregnancy. Cyp11a1 in a bacterial artificial chromosome was injected into mouse embryos to generate transgenic mice with transgene expression that recapitulated endogenous Cyp11a1 expression. Cyp11a1 transgenic females displayed reduced pregnancy rate, impaired implantation and placentation, and decreased litter size in utero, although they produced comparable numbers of blastocysts. The differentiation of transgenic luteal cells was delayed during early pregnancy as shown by the delayed activation of genes involved in steroidogenesis and cholesterol availability. Luteal cell mitochondria were elongated, and their numbers were reduced, with morphology and numbers similar to those observed in granulosa cells. Transgenic luteal cells accumulated lipid droplets and secreted less progesterone during early pregnancy. The progesterone level returned to normal on gestation day 9 but was not properly withdrawn at term, leading to delayed stillbirth. P4 supplementation rescued the implantation rates but not the ovarian defects. Thus, overexpression of Cyp11a1 disrupts normal development of the corpus luteum, leading to progesterone insufficiency during early pregnancy. Misregulation of the progesterone production in Cyp11a1 transgenic mice during pregnancy resulted in aberrant implantation, anomalous placentation, and delayed parturition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1095/biolreprod.113.110833DOI Listing
October 2013

Selective inhibition of the NLRP3 inflammasome by targeting to promyelocytic leukemia protein in mouse and human.

Blood 2013 Apr 21;121(16):3185-94. Epub 2013 Feb 21.

Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan.

The functional activities of the tumor suppressor promyelocytic leukemia protein (PML) are mostly associated with its nuclear location. In the present study, we discovered an unexpected role of PML in NLRP3 inflammasome activation. In PML-deficient macrophages, the production of IL-1β was strongly impaired. The expression of pro-IL-1β, NLRP3, ASC, and procaspase-1 was not affected in Pml(-/-) macrophages. PML deficiency selectively reduced the processing of procaspase-1. We further showed that PML is required for the assembly of the NLRP3 inflammasome in reconstitution experiment. All PML isoforms were capable of stimulating NLRP3 inflammasome activation. In Pml(-/-) macrophages, the generation of reactive oxygen species and release of mitochondrial DNA were decreased. The involvement of PML in inflammasome activation constitutes an important activity of PML and reveals a new mechanism underlying the inflammasome activation. In addition, downregulation of PML by arsenic trioxide suppressed monosodium urate (MSU)-induced IL-1β production, suggesting that targeting to PML could be used to treat NLRP3 inflammasome-associated diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2012-05-432104DOI Listing
April 2013

Impaired phosphorylation of Na(+)-K(+)-2Cl(-) cotransporter by oxidative stress-responsive kinase-1 deficiency manifests hypotension and Bartter-like syndrome.

Proc Natl Acad Sci U S A 2011 Oct 4;108(42):17538-43. Epub 2011 Oct 4.

Division of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan.

Na(+)-K(+)-2Cl(-) cotransporters (NKCCs), including NKCC1 and renal-specific NKCC2, and the Na(+)-Cl(-) cotransporter (NCC) play pivotal roles in the regulation of blood pressure (BP) and renal NaCl reabsorption. Oxidative stress-responsive kinase-1 (OSR1) is a known upstream regulator of N(K)CCs. We generated and analyzed global and kidney tubule-specific (KSP) OSR1 KO mice to elucidate the physiological role of OSR1 in vivo, particularly on BP and kidney function. Although global OSR1(-/-) mice were embryonically lethal, OSR1(+/-) mice had low BP associated with reduced phosphorylated (p) STE20 (sterile 20)/SPS1-related proline/alanine-rich kinase (SPAK) and p-NKCC1 abundance in aortic tissue and attenuated p-NKCC2 abundance with increased total and p-NCC expression in the kidney. KSP-OSR1(-/-) mice had normal BP and hypercalciuria and maintained significant hypokalemia on a low-K(+) diet. KSP-OSR1(-/-) mice exhibited impaired Na(+) reabsorption in the thick ascending loop on a low-Na(+) diet accompanied by remarkably decreased expression of p-NKCC2 and a blunted response to furosemide, an NKCC2 inhibitor. The expression of total SPAK and p-SPAK was significantly increased in parallel to that of total NCC and p-NCC despite unchanged total NKCC2 expression. These results suggest that, globally, OSR1 is involved in the regulation of BP and renal tubular Na(+) reabsorption mainly via the activation of NKCC1 and NKCC2. In the kidneys, NKCC2 but not NCC is the main target of OSR1 and the reduced p-NKCC2 in KSP-OSR1(-/-) mice may lead to a Bartter-like syndrome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1107452108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198379PMC
October 2011

Different NK cell developmental events require different levels of IL-15 trans-presentation.

J Immunol 2011 Aug 29;187(3):1212-21. Epub 2011 Jun 29.

Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan.

NK cell development requires IL-15, which is "trans-presented" to IL-15Rβγ on NK cells by IL-15Rα on other cells. In this study, we report that different levels of IL-15 trans-presentation are required for different NK cell developmental events to reach full maturation status. Because the IL-15Rα intracellular domain has the capacity to recruit signaling molecules, we generated knockin and transgenic (Tg) mice that lack the intracellular domain to assess the role of the IL-15 trans-presentation level independent of the function of this domain. The level of IL-15Rα on various cells of these mice follows the order WT > Tg6 > knockin > Tg1 ≥ knockout. Bone marrow (BM)-derived dendritic cells prepared from these mice induced Stat5 phosphorylation in NK cells. The level of phospho-Stat5 correlated with the level of IL-15Rα on BMDCs, thus offering the opportunity to study quantitative effects of IL-15 trans-presentation on NK cell development in vivo. We found that NK cell homeostasis, mature NK cell differentiation, and acquisition of Ly49 receptor and effector functions require different levels of IL-15 trans-presentation input to achieve full status. All NK cell developmental events examined were quantitatively regulated by the IL-15Rα level of BM-derived and radiation-resistant accessory cells, but not by IL-15Rα of NK cells. We also found that IL-15Rα of radiation-resistant cells was more potent than IL-15Rα of BM-derived accessory cells in support of stage 2 to stage 3 splenic mNK differentiation. In summary, each examined developmental event required a particular level of IL-15 trans-presentation by accessory cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.4049/jimmunol.1100331DOI Listing
August 2011

Sarm1, a negative regulator of innate immunity, interacts with syndecan-2 and regulates neuronal morphology.

J Cell Biol 2011 May 9;193(4):769-84. Epub 2011 May 9.

Institute of Molecular Biology and 2 Molecular and Cell Biology Program, Taiwan International Graduate Program, Graduate Institute of Life Sciences, National Defense Medical Center and Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan.

Dendritic arborization is a critical neuronal differentiation process. Here, we demonstrate that syndecan-2 (Sdc2), a synaptic heparan sulfate proteoglycan that triggers dendritic filopodia and spine formation, regulates dendritic arborization in cultured hippocampal neurons. This process is controlled by sterile α and TIR motif-containing 1 protein (Sarm1), a negative regulator of Toll-like receptor 3 (TLR3) in innate immunity signaling. We show that Sarm1 interacts with and receives signal from Sdc2 and controls dendritic arborization through the MKK4-JNK pathway. In Sarm1 knockdown mice, dendritic arbors of neurons were less complex than those of wild-type littermates. In addition to acting downstream of Sdc2, Sarm1 is expressed earlier than Sdc2, which suggests that it has multiple roles in neuronal morphogenesis. Specifically, it is required for proper initiation and elongation of dendrites, axonal outgrowth, and neuronal polarization. These functions likely involve Sarm1-mediated regulation of microtubule stability, as Sarm1 influenced tubulin acetylation. This study thus reveals the molecular mechanism underlying the action of Sarm1 in neuronal morphogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201008050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3166868PMC
May 2011

Progressive renal distortion by multiple cysts in transgenic mice expressing artificial microRNAs against Pkd1.

J Pathol 2010 Nov;222(3):238-48

Department of Life Sciences, National Taiwan Normal University, Taipei, Taiwan, Republic of China.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common life-threatening inherited diseases, and the PKD1 gene is responsible for most cases of this disease. Previous efforts to establish a mouse model that recapitulates the phenotypic characteristics of ADPKD, which have used conventional or conditional knockout of the mouse orthologue Pkd1, have been unsuccessful or unreliable. In a previous study, we described the generation of a novel Pkd1 hypomorphic allele, in which Pkd1 expression was significantly reduced but not totally blocked. These Pkd1 homozygous mutant mice rapidly developed renal cystic disease, supporting the hypothesis that 'haploinsufficiency' explains development of the ADPKD phenotype. In the present study, we further investigated the Pkd1 haploinsufficiency effect by generating Pkd1 knockdown transgenic mice with co-cistronic expression of two miRNA hairpins specific to Pkd1 transcript and an Emerald GFP reporter driven by a human ubiquitin B promoter. Two transgenic lines which had ∼60-70% reduction of Pkd1 expression developed severe renal cystic disease at a rate similar to that of human ADPKD. These results further support the haploinsufficiency hypothesis, and suggest that the onset and progression of the renal cystic diseases are correlated with the level of Pkd1 expression. The two novel mutant lines of mice appear to be ideal models for the study of ADPKD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/path.2765DOI Listing
November 2010

Impaired water reabsorption in mice deficient in the type VI adenylyl cyclase (AC6).

FEBS Lett 2010 Jul 11;584(13):2883-90. Epub 2010 May 11.

Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.

Adenylyl cyclase (AC) type VI (AC6) is a calcium-inhibitable enzyme which produces cAMP upon stimulation. Herein, we characterized the specific role of AC6 in the kidneys using two AC6-knockout mouse lines. Immunohistochemical staining revealed that AC6 exists in the tubular parts of the nephron and collecting duct. Activities of AC evoked by forskolin or a selective agonist of the V2 vasopressin receptor were lower in the kidneys of AC6-null mice compared to those of wildtype mice. Results of a metabolic cage assay and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) showed for the first time that AC6 plays a critical role in regulating water homeostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.febslet.2010.05.004DOI Listing
July 2010

Mouse kidney progenitor cells accelerate renal regeneration and prolong survival after ischemic injury.

Stem Cells 2010 Mar;28(3):573-84

Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

Acute tubular necrosis is followed by regeneration of damaged renal tubular epithelial cells, and renal stem cells are supposed to contribute to this process. The purpose of our study is to test the hypothesis that renal stem cells isolated from adult mouse kidney accelerate renal regeneration via participation in the repair process. A unique population of cells exhibiting characteristics consistent with renal stem cells, mouse kidney progenitor cells (MKPC), was isolated from Myh9 targeted mutant mice. Features of these cells include (1) spindle-shaped morphology, (2) self-renewal of more than 100 passages without evidence of senescence, and (3) expression of Oct-4, Pax-2, Wnt-4, WT-1, vimentin, alpha-smooth muscle actin, CD29, and S100A4 but no SSEA-1, c-kit, or other markers of more differentiated cells. MKPC exhibit plasticity as demonstrated by the ability to differentiate into endothelial cells and osteoblasts in vitro and endothelial cells and tubular epithelial cells in vivo. The origin of the isolated MKPC was from the interstitium of medulla and papilla. Importantly, intrarenal injection of MKPC in mice with ischemic injury rescued renal damage, as manifested by decreases in peak serum urea nitrogen, the infarct zone, and the necrotic injury. Seven days after the injury, some MKPC formed vessels with red blood cells inside and some incorporated into renal tubules. In addition, MKPC treatment reduces the mortality in mice after ischemic injury. Our results indicate that MKPC represent a multipotent adult stem cell population, which may contribute to the renal repair and prolong survival after ischemic injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.310DOI Listing
March 2010

TDP-43, a neuro-pathosignature factor, is essential for early mouse embryogenesis.

Genesis 2010 Jan;48(1):56-62

Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.

TDP-43 is a highly conserved and ubiquitously expressed nuclear protein. It has been implicated in the regulation of transcription, alternative splicing, translation, and neuronal plasticity. TDP-43 has also been shown to be a disease signature protein associated with several neurodegenerative diseases including amyotrophic lateral sclerosis. However, the correlation of the physiological functions of TDP-43 with these diseases remains unknown. We have used the gene targeting approach to disrupt the expression of TDP-43 in mouse. Loss of the TDP-43 expression results in peri-implantation lethality of mice between embryonic days (E) 3.5 and 6.5. Blastocysts of the homozygous Tardbp null mutants are morphologically normal, but exhibit defective outgrowth of the inner cell mass in vitro. Our data demonstrate the essential function of TDP-43 in peri-implantation stage during the embryo development, likely because of its involvement in multiple biological processes in a variety of cell types.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/dvg.20584DOI Listing
January 2010

Deltex1 is a target of the transcription factor NFAT that promotes T cell anergy.

Immunity 2009 Jul;31(1):72-83

Graduate Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 11221, Taiwan, ROC.

The molecular process underlying T cell anergy is incompletely understood. Deltex1 (DTX1) is a Notch target with unknown physiological function. Here we show that Dtx1 was a transcription target of nuclear factor of activated T cells (NFAT) and participated in T cell anergy. DTX1 protein was upregulated during T cell anergy, and transgenic expression of Dtx1 attenuated T cell activation. DTX1 inhibited T cell activation by both E3-dependent and E3-independent mechanisms. In addition, DTX1 suppressed T cell activation in the absence of its Notch-binding domain. Importantly, DTX1 regulated the expression of two anergy-associated molecules, growth arrest and DNA-damage-inducible 45 beta (Gadd45 beta) and Cbl-b. DTX1 interacted with early growth response 2 (Egr-2) for optimum expression of Cbl-b. Furthermore, deficiency of DTX1 augmented T cell activation, conferred resistance to anergy induction, enhanced autoantibody generation, and increased inflammation. DTX1 therefore represents a component downstream of calcium-NFAT signaling that regulates T cell anergy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2009.04.017DOI Listing
July 2009

Essential role of nephrocystin in photoreceptor intraflagellar transport in mouse.

Hum Mol Genet 2009 May 9;18(9):1566-77. Epub 2009 Feb 9.

Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.

Nephrocystin mutations account for the vast majority of juvenile nephronophthisis, the most common inherited cause of renal failure in children. Nephrocystin has been localized to the ciliary transition zone of epithelial cells or its analogous structure, connecting cilium of retinal photoreceptors. Thus, the retinal degeneration associated with nephronophthisis may be explained by a functional ciliary defect. However, the function of nephrocystin in cilium assembly and maintenance of common epithelial cells and photoreceptors is still obscure. Here, we used Nphp1-targeted mutant mice and transgenic mice expressing EmGFP-tagged nephrocystin to demonstrate that nephrocystin located at connecting cilium axoneme can affect the sorting mechanism and transportation efficiency of the traffic machinery between inner and outer segments of photoreceptors. This traffic machinery is now recognized as intraflagellar transport (IFT); a microtubule-based transport system consisting of motors, IFT particles and associated cargo molecules. Nephrocystin seems to control some of the IFT particle components moving along the connecting cilia so as to regulate this inter-segmental traffic. Our novel findings provide a clue to unraveling the regulatory mechanism of nephrocystin in IFT machinery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddp068DOI Listing
May 2009

Targeted disruption of Nphp1 causes male infertility due to defects in the later steps of sperm morphogenesis in mice.

Hum Mol Genet 2008 Nov 5;17(21):3368-79. Epub 2008 Aug 5.

Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan.

Juvenile nephronophthisis type I is the most common genetic disorder causing end-stage renal failure in children and young adults. The defective gene responsible has been identified as NPHP1. Its gene product, nephrocystin-1, is a novel protein of uncertain function that is widely expressed in many tissues and not just confined to the kidney. To gain insight into the physiological function of nephrocystin, Nphp1-targeted mutant mice were generated by homologous recombination. Interestingly, homozygous Nphp1 mutant mice were viable without renal manifestations of nephronophthisis. They appeared normal, but males were infertile with oligoteratozoospermia. Histological analysis of the seminiferous tubules showed that spermatogenesis was blocked at the early stages of spermatid elongation, with degenerating spermatids sloughing off into the lumen. Electron microscopic analysis revealed detachment of early elongating spermatids from Sertoli cells, and a failure of sperm head and tail morphogenesis. However, a few mature spermatozoa were still deposited in the epididymis, though they were frequently dead, immotile, or malformed. These novel findings indicate that nephrocystin is critically required for the differentiation of early elongating spermatids into spermatozoa in mice. The possible roles of nephrocystin in the formation and maintenance of Sertoli-spermatid junctions are still under investigation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddn231DOI Listing
November 2008