Impact of translational regulation on diel expression revealed by time-series ribosome profiling in Arabidopsis.

Plants have developed the ability to adjust to the day/night cycle through the expression of diel genes, which allow them to effectively respond to environmental changes and optimise their growth and development. Diel oscillations also have substantial implications in many physiological processes, including photosynthesis, floral development, and environmental stress responses. The expression of diel genes is regulated by a combination of the circadian clock and responses to environmental cues, such as light and temperature. A great deal of information is available on the transcriptional regulation of diel gene expression. However, the extent to which translational regulation is involved in controlling diel changes in expression is not yet clear. To investigate the impact of translational regulation on diel expression, we conducted Ribo-seq and RNA-seq analyses on a time-series sample of Arabidopsis shoots cultivated under a 12 h light/dark cycle. Our results showed that translational regulation is involved in about 71% of the genes exhibiting diel changes in mRNA abundance or translational activity, including clock genes, many of which are subject to both translational and transcriptional control. They also revealed that the diel expression of glycosylation and ion-transporter-related genes is mainly established through translational regulation. The expression of several diel genes likely subject to translational regulation through upstream open-reading frames was also determined.

Phylogeny-linked occurrence of ribosome stalling on the mRNAs of Arabidopsis unfolded protein response factor bZIP60 orthologs in divergent plant species.

The bZIP60, XBP1 and HAC1 mRNAs encode transcription factors that mediate the unfolded protein response (UPR) in plants, animals and yeasts, respectively. Upon UPR, these mRNAs undergo unconventional cytoplasmic splicing on the endoplasmic reticulum (ER) to produce active transcription factors. Although cytoplasmic splicing is conserved, the ER targeting mechanism differs between XBP1 and HAC1. The ER targeting of HAC1 mRNA occurs before translation, whereas that of XBP1 mRNA involves a ribosome-nascent chain complex that is stalled when a hydrophobic peptide emerges from the ribosome; the corresponding mechanism is unknown for bZIP60. Here, we analyzed ribosome stalling on bZIP60 orthologs of plants. Using a cell-free translation system, we detected nascent peptide-mediated ribosome stalling during the translation elongation of the mRNAs of Arabidopsis, rice and Physcomitrium (moss) orthologs, and the termination-step stalling in the Selaginella (lycopod) ortholog, all of which occurred ∼50 amino acids downstream of a hydrophobic region. Transfection experiments showed that ribosome stalling contributes to cytoplasmic splicing in bZIP60u orthologs of Arabidopsis and Selaginella. In contrast, ribosome stalling was undetectable for liverwort, Klebsormidium (basal land plant), and green algae orthologs. This study highlights the evolutionary diversity of ribosome stalling and its contribution to ER targeting in plants.

Alterations in choroidal circulatory dynamics and choroidal thickness before and after treatment in posterior scleritis.

BACKGROUND: Posterior scleritis is an inflammatory reaction of the sclera that occurs posterior to the ora serrata. The aim of this study was to present a case of posterior scleritis and to analyze choroidal circulatory and structural changes using laser speckle flowgraphy (LSFG) and optical coherence tomography (OCT), respectively. CASE PRESENTATION: A 64-year-old man presented to our department because of hyperemia of the left eye for one week, diplopia, ocular pain, and distorted vision when looking leftward. At an initial examination, his best-corrected visual acuity was 1.0 Oculi uterque (OU), with mild conjunctival hyperemia oculus dexter (OD) and marked ciliary hyperemia oculus sinister (OS). Color fundus photographs revealed a cluster of choroidal folds extending from the macula to the inferior retinal region OS. Swept-Source OCT showed choroidal thickening OD, and bacillary layer detachment and paracentral middle maculopathy on the paracentral side of the optic nerve papilla, suggesting severe inflammation. Fluorescein angiography showed hyperfluorescence in the optic disc and window defects around the macula OU. Indocyanine green angiography showed mottled choroidal vascular hyperpermeability findings in the late stage. B-mode echography displayed thickening of the posterior wall of the left eye. Orbital magnetic resonance imaging showed the thickened posterior eyeball. The patient was diagnosed with posterior scleritis, and 30 mg of oral prednisolone was then given and tapered off over the next 4 months. The hyperemia and intraocular inflammation resolved after the treatment. The rate of change in macular blood flow assessed by the mean blur rate on LSFG was 20.5% and 20.2% decrease OD and OS, respectively, before and after treatment. The central choroidal thickness showed 8.8% and 37.8% decrease OD and OS, respectively. CONCLUSION: Posterior scleritis complicated with choroiditis was suggested to show different choroidal circulatory dynamics from those in other choroidal inflammations.

Genome-wide identification of Arabidopsis non-AUG-initiated upstream ORFs with evolutionarily conserved regulatory sequences that control protein expression levels.

KEY MESSAGE: This study identified four novel regulatory non-AUG-initiated upstream ORFs (uORFs) with evolutionarily conserved sequences in Arabidopsis and elucidated the mechanism by which a non-AUG-initiated uORF promotes main ORF translation. Upstream open reading frames (uORFs) are short ORFs found in the 5'-untranslated regions (5'-UTRs) of eukaryotic transcripts and can influence the translation of protein-coding main ORFs (mORFs). Recent genome-wide ribosome profiling studies have revealed that hundreds or thousands of uORFs initiate translation at non-AUG start codons. However, the physiological significance of these non-AUG uORFs has so far been demonstrated for only a few of them. In this study, to identify physiologically important regulatory non-AUG uORFs in Arabidopsis, we took an approach that combined bioinformatics and experimental analysis. Since physiologically important non-AUG uORFs are likely to be conserved across species, we first searched the Arabidopsis genome for non-AUG-initiated uORFs with evolutionarily conserved sequences. Then, we examined the effects of the conserved non-AUG uORFs on the expression of the downstream mORFs using transient expression assays. As a result, three inhibitory and one promotive non-AUG uORFs were identified. Among the inhibitory non-AUG uORFs, two exerted repressive effects on mORF expression in an amino acid sequence-dependent manner. These two non-AUG uORFs are likely to encode regulatory peptides that cause ribosome stalling, thereby enhancing their repressive effects. In contrast, one of the identified regulatory non-AUG uORFs promoted mORF expression by alleviating the inhibitory effect of a downstream AUG-initiated uORF. These findings provide insights into the mechanisms that enable non-AUG uORFs to play regulatory roles despite their low translation initiation efficiencies.

Upstream open reading frame-mediated upregulation of ANAC082 expression in response to nucleolar stress in Arabidopsis.

Perturbations in ribosome biogenesis cause a type of cellular stress called nucleolar or ribosomal stress, which triggers adaptive responses in both animal and plant cells. The Arabidopsis ANAC082 transcription factor has been identified as a key mediator of the plant nucleolar stress response. The 5'-untranslated region (5'-UTR) of ANAC082 mRNA contains an upstream ORF (uORF) encoding an evolutionarily conserved amino acid sequence. Here, we report that this uORF mediates the upregulation of ANAC082 expression in response to nucleolar stress. When transgenic Arabidopsis plants containing a luciferase reporter gene under the control of the ANAC082 promoter and 5'-UTR were treated with reagents that induced nucleolar stress, expression of the reporter gene was enhanced in a uORF sequence-dependent manner. Additionally, we examined the effect of an endoplasmic reticulum (ER) stress-inducing reagent on reporter gene expression because the closest homolog of ANAC082 in Arabidopsis, ANAC103, is involved in the ER stress response. However, the ANAC082 uORF did not respond to ER stress. Interestingly, although ANAC103 has a uORF with an amino acid sequence similar to that of the ANAC082 uORF, the C-terminal sequence critical for regulation is not well conserved among ANAC103 homologs in Brassicaceae. Transient expression assays revealed that unlike the ANAC082 uORF, the ANAC103 uORF does not exert a sequence-dependent repressive effect. Altogether, our findings suggest that the ANAC082 uORF is important for the nucleolar stress response but not for the ER stress response, and that for this reason, the uORF sequence-dependent regulation was lost in ANAC103 during evolution.

Translational Landscape of a C4 Plant, Sorghum bicolor, Under Normal and Sulfur-Deficient Conditions.

Recent accumulation of genomic and transcriptomic information has facilitated genetic studies. Increasing evidence has demonstrated that translation is an important regulatory step, and the transcriptome does not necessarily reflect the profile of functional protein production. Deep sequencing of ribosome-protected mRNA fragments (ribosome profiling or Ribo-seq) has enabled genome-wide analysis of translation. Sorghum is a C4 cereal important not only as food but also as forage and a bioenergy resource. Its resistance to harsh environments has made it an agriculturally important research subject. Yet genome-wide translational profiles in sorghum are still missing. In this study, we took advantage of Ribo-seq and identified actively translated reading frames throughout the genome. We detected translation of 4,843 main open reading frames (ORFs) annotated in the sorghum reference genome version 3.1 and revealed a number of unannotated translational events. A comparison of the transcriptome and translatome between sorghums grown under normal and sulfur-deficient conditions revealed that gene expression is modulated independently at transcript and translation levels. Our study revealed the translational landscape of sorghum's response to sulfur and provides datasets that could serve as a fundamental resource to extend genetic research on sorghum, including studies on translational regulation.

Imbalanced choroidal circulation in eyes with asymmetric dilated vortex vein.

PURPOSE: Asymmetric dilated vortex vein (ADVV) observed in eyes with pachychoroid spectrum diseases is thought to be due to congestion of choroidal blood flow. The purpose of this study was to quantitatively investigate the blood flow velocity of ADVV using laser speckle flowgraphy (LSFG). STUDY DESIGN: Retrospective case series. METHODS: This was a retrospective case series with 23 eyes of 18 patients with ADVV on en-face OCT. A pair of choroidal veins from ADVV side (defined as ADVV vein) and non-ADVV side (defined as non-ADVV vein) was selected in each eye under the following criteria: (i) equivalent proximity to the deviated watershed, (ii) does not overlap with retinal blood vessels in the en-face OCT image, (iii) has approximately the same blood vessel diameter. Rubber bands were placed on the selected choroidal veins on the LSFG color map. Mean blur rate (MBR) values of ADVV and non-ADVV veins were statistically compared. RESULTS: The average MBR was 10.11 ± 1.9 in the ADVV veins and 13.49 ± 6.2 in the non-ADVV veins, showing significantly lower values in the ADVV veins (P = 0.03). The blood vessel diameter of the ADVV was 10.26 ± 3.0 and in the non-ADVV veins, 10.63 ± 2.9 pixels; not significantly different (P = 0.66). The distance from the deviated watershed to the ADVV was 53.3 ± 24.8 and to the non-ADVV veins, 46.80 ± 20.3 pixels; not significantly different (P = 0.41). CONCLUSION: In eyes with ADVV, the blood flow velocity in the ADVV veins was lower than in the non-ADVV veins, suggesting anatomical congestion of ADVV.

Optic Nerve Head Microcirculation in Eyes with Vogt-Koyanagi-Harada Disease Accompanied by Anterior Ischemic Optic Neuropathy.

Anterior ischemic optic neuropathy (AION) is infrequently complicated with Vogt-Koyanagi-Harada (VKH) disease. We quantitatively examined sequential changes in the morphology and circulation hemodynamics, using a C-scan of optical coherence tomography (OCT) and laser speckle flowgraphy (LSFG) in a patient with VKH disease accompanied by AION. A 65-year-old female complained of blurred vision in both of her eyes. The patient presented with optic disc swelling and remarkable choroidal thickening detected by OCT bilaterally. The diagnosis of VKH disease was established based on the presence of pleocytosis detected in the cerebrospinal fluid and hypofluorescent dark dots scattered all around the fundus, detected by indocyanine green angiography. Goldmann perimetry detected visual field defects, similar to superior altitudinal hemianopsia in the right eye and similar to inferior altitudinal hemianopsia in the left eye. The patient was suspected to have developed AION in both eyes. The patient received methylprednisolone pulse therapy, followed by oral prednisolone. With these treatments, the optic disc swelling disappeared. However, optic disc atrophy with visual field defects remained in both eyes. An OCT C-scan showed the ganglion cell complex (GCC) and circumpapillary retinal nerve fiber layer (cpRNFL) thickness getting thinner below the normal range, and LSFG showed the decrease in optic nerve head (ONH) tissue microcirculation. These results supported the occurrence of AION in this patient with VKH disease. The analysis of GCC and cpRNFL thickness and ONH microcirculation would be useful for supporting the occurrence of AION in a case of VKH disease.

Global analysis of boron-induced ribosome stalling reveals its effects on translation termination and unique regulation by AUG-stops in Arabidopsis shoots.

We previously reported that ribosome stalling at AUG-stop sequences in the 5'-untranslated region plays a critical role in regulating the expression of Arabidopsis thaliana NIP5;1, which encodes a boron uptake transporter, in response to boron conditions in media. This ribosome stalling is triggered specifically by boric acid, but the mechanisms are unknown. Although upstream open reading frames (uORFs) are known in many cases to regulate translation through peptides encoded by the uORF, AUG-stop stalling does not involve any peptide synthesis. The unique feature of AUG-stops - that termination follows immediately after initiation - suggests a possible effect of boron on the translational process itself. However, the generality of AUG-stop-mediated translational regulation and the effect of boron on translation at the genome scale are not clear. Here, we conducted a ribosome profiling analysis to reveal the genome-wide regulation of translation in response to boron conditions in A. thaliana shoots. We identified hundreds of translationally regulated genes that function in various biological processes. Under high-boron conditions, transcripts with reduced translation efficiency were rich in uORFs, highlighting the importance of uORF-mediated translational regulation. We found 673 uORFs that had more frequent ribosome association. Moreover, transcripts that were translationally downregulated under high-boron conditions were rich in minimum uORFs (AUG-stops), suggesting that AUG-stops play a global role in the boron response. Metagene analysis revealed that boron increased the ribosome occupancy of stop codons, indicating that this element is involved in global translational termination processes.

Optineurin defects cause TDP43-pathology with autophagic vacuolar formation.

We previously showed that optineurin (OPTN) mutations lead to the development of amyotrophic lateral sclerosis. The association between OPTN mutations and the pathogenesis of amyotrophic lateral sclerosis remains unclear. To investigate the mechanism underlying its pathogenesis, we generated Optn knockout mice. We evaluated histopathological observations of these mice and compared with those of OPTN- amyotrophic lateral sclerosis cases to investigate the mechanism underlying the pathogenesis of amyotrophic lateral sclerosis caused by OPTN mutations. The Optn (-/-) mice presented neuronal autophagic vacuoles immunopositive for charged multivesicular body protein 2b, one of the hallmarks of granulovacuolar degenerations, in the cytoplasm of spinal cord motor neurons at the age of 8 months and the OPTN- amyotrophic lateral sclerosis case with homozygous Q398X mutation. In addition, Optn (-/-) mice showed TAR-DNA binding protein 43/sequestosome1/p62 -positive cytoplasmic inclusions and the clearance of nuclear TAR-DNA binding protein 43. The axonal degeneration of the sciatic nerves was observed in Optn (-/-) mice. However, we could not observe significant differences in survival time, body weight, and motor functions, at 24 months. Our findings suggest that homozygous OPTN deletion or mutations might result in autophagic dysfunction and TAR-DNA binding protein 43 mislocalization, thereby leading to neurodegeneration of motor neurons. These findings indicate that the Optn (-/-) mice recapitulate both common and specific pathogenesis of amyotrophic lateral sclerosis associated with autophagic abnormalities. Optn (-/-) mice could serve as a mouse model for the development of therapeutic strategies.

Comprehensive genome-wide identification of angiosperm upstream ORFs with peptide sequences conserved in various taxonomic ranges using a novel pipeline, ESUCA.

BACKGROUND: Upstream open reading frames (uORFs) in the 5'-untranslated regions (5'-UTRs) of certain eukaryotic mRNAs encode evolutionarily conserved functional peptides, such as cis-acting regulatory peptides that control translation of downstream main ORFs (mORFs). For genome-wide searches for uORFs with conserved peptide sequences (CPuORFs), comparative genomic studies have been conducted, in which uORF sequences were compared between selected species. To increase chances of identifying CPuORFs, we previously developed an approach in which uORF sequences were compared using BLAST between Arabidopsis and any other plant species with available transcript sequence databases. If this approach is applied to multiple plant species belonging to phylogenetically distant clades, it is expected to further comprehensively identify CPuORFs conserved in various plant lineages, including those conserved among relatively small taxonomic groups. RESULTS: To efficiently compare uORF sequences among many species and efficiently identify CPuORFs conserved in various taxonomic lineages, we developed a novel pipeline, ESUCA. We applied ESUCA to the genomes of five angiosperm species, which belong to phylogenetically distant clades, and selected CPuORFs conserved among at least three different orders. Through these analyses, we identified 89 novel CPuORF families. As expected, ESUCA analysis of each of the five angiosperm genomes identified many CPuORFs that were not identified from ESUCA analyses of the other four species. However, unexpectedly, these CPuORFs include those conserved across wide taxonomic ranges, indicating that the approach used here is useful not only for comprehensive identification of narrowly conserved CPuORFs but also for that of widely conserved CPuORFs. Examination of the effects of 11 selected CPuORFs on mORF translation revealed that CPuORFs conserved only in relatively narrow taxonomic ranges can have sequence-dependent regulatory effects, suggesting that most of the identified CPuORFs are conserved because of functional constraints of their encoded peptides. CONCLUSIONS: This study demonstrates that ESUCA is capable of efficiently identifying CPuORFs likely to be conserved because of the functional importance of their encoded peptides. Furthermore, our data show that the approach in which uORF sequences from multiple species are compared with those of many other species, using ESUCA, is highly effective in comprehensively identifying CPuORFs conserved in various taxonomic ranges.

Reverse genetics-based biochemical studies of the ribosomal exit tunnel constriction region in eukaryotic ribosome stalling: spatial allocation of the regulatory nascent peptide at the constriction.

A number of regulatory nascent peptides have been shown to regulate gene expression by causing programmed ribosome stalling during translation. Nascent peptide emerges from the ribosome through the exit tunnel, and one-third of the way along which ß-loop structures of ribosomal proteins uL4 and uL22 protrude into the tunnel to form the constriction region. Structural studies have shown interactions between nascent peptides and the exit tunnel components including the constriction region. In eukaryotes, however, there is a lack of genetic studies for the involvement of the constriction region in ribosome stalling. Here, we established transgenic Arabidopsis lines that carry mutations in the ß-loop structure of uL4. Translation analyses using a cell-free translation system derived from the transgenic Arabidopsis carrying the mutant ribosome showed that the uL4 mutations reduced the ribosome stalling of four eukaryotic stalling systems, including those for which stalled structures have been solved. Our data, which showed differential effects of the uL4 mutations depending on the stalling systems, explained the spatial allocations of the nascent peptides at the constriction that were deduced by structural studies. Conversely, our data may predict allocation of the nascent peptide at the constriction of stalling systems for which structural studies are not done.

Publisher Correction: Structure and function of Vms1 and Arb1 in RQC and mitochondrial proteome homeostasis.

Change history: In this Letter, the bottom blot in Fig. 2g (for 'IB: Myc') was missing. This has been corrected online.

Structure and function of Vms1 and Arb1 in RQC and mitochondrial proteome homeostasis.

Ribosome-associated quality control (RQC) provides a rescue pathway for eukaryotic cells to process faulty proteins after translational stalling of cytoplasmic ribosomes1-6. After dissociation of ribosomes, the stalled tRNA-bound peptide remains associated with the 60S subunit and extended by Rqc2 by addition of C-terminal alanyl and threonyl residues (CAT tails)7-9, whereas Vms1 catalyses cleavage and release of the peptidyl-tRNA before or after addition of CAT tails10-12. In doing so, Vms1 counteracts CAT-tailing of nuclear-encoded mitochondrial proteins that otherwise drive aggregation and compromise mitochondrial and cellular homeostasis13. Here we present structural and functional insights into the interaction of Saccharomyces cerevisiae Vms1 with 60S subunits in pre- and post-peptidyl-tRNA cleavage states. Vms1 binds to 60S subunits with its Vms1-like release factor 1 (VLRF1), zinc finger and ankyrin domains. VLRF1 overlaps with the Rqc2 A-tRNA position and interacts with the ribosomal A-site, projecting its catalytic GSQ motif towards the CCA end of the tRNA, its Y285 residue dislodging the tRNA A73 for nucleolytic cleavage. Moreover, in the pre-state, we found the ABCF-type ATPase Arb1 in the ribosomal E-site, which stabilizes the delocalized A73 of the peptidyl-tRNA and stimulates Vms1-dependent tRNA cleavage. Our structural analysis provides mechanistic insights into the interplay of the RQC factors Vms1, Rqc2 and Arb1 and their role in the protection of mitochondria from the aggregation of toxic proteins.

The Pro-Oxidant Activity of Pheomelanin is Significantly Enhanced by UVA Irradiation: Benzothiazole Moieties Are More Reactive than Benzothiazine Moieties.

It is generally considered that eumelanin (EM) is photoprotective while pheomelanin (PM) is phototoxic. A recent study using a mouse model demonstrated that PM produces reactive oxygen species (ROS) that cause DNA damage and eventually lead to melanomagenesis. A biochemical study showed that PM possesses a pro-oxidant activity. PM consists of benzothiazine (BT) and benzothiazole (BZ) moieties, BT moieties being transformed to BZ moieties by heat or light. In this study, we compared the effects of ultraviolet A (UVA) irradiation using synthetic PMs with different BT to BZ ratios and using various coat color mouse hairs. We found that UVA irradiation of BZ-PM increased glutathione (GSH) depletion and generated more H2O2 than UVA irradiation of BT-PM. Non-irradiated controls did not exhibit strong pro-oxidant activities. Upon UVA irradiation, yellow mouse hairs oxidized GSH and produced H2O2 faster than black or albino mouse hairs. Next, to examine the mechanism of the pro-oxidant activity of BT-PM and BZ-PM, we examined the pro-oxidant activities of 7-(2-amino-2-carboxyethyl)-dihydro-1,4-benzothiazine-3-carboxylic acid (DHBTCA) and 6-(2-amino-2-carboxyethyl)-4-hydroxybenzothiazole (BZ-AA) as BT and BZ monomers, respectively. Their pro-oxidant activities were similar, but a large difference was seen in the effects of ROS scavengers, which suggests that the redox reactions may proceed via singlet oxygen in BZ-AA and via superoxide anions in DHBTCA. These results show that UVA enhances the pro-oxidant activity of PM, in particular BZ-PM.

Reactive oxygen species upregulate expression of muscle atrophy-associated ubiquitin ligase Cbl-b in rat L6 skeletal muscle cells.

Unloading-mediated muscle atrophy is associated with increased reactive oxygen species (ROS) production. We previously demonstrated that elevated ubiquitin ligase casitas B-lineage lymphoma-b (Cbl-b) resulted in the loss of muscle volume (Nakao R, Hirasaka K, Goto J, Ishidoh K, Yamada C, Ohno A, Okumura Y, Nonaka I, Yasutomo K, Baldwin KM, Kominami E, Higashibata A, Nagano K, Tanaka K, Yasui N, Mills EM, Takeda S, Nikawa T. Mol Cell Biol 29: 4798-4811, 2009). However, the pathological role of ROS production associated with unloading-mediated muscle atrophy still remains unknown. Here, we showed that the ROS-mediated signal transduction caused by microgravity or its simulation contributes to Cbl-b expression. In L6 myotubes, the assessment of redox status revealed that oxidized glutathione was increased under microgravity conditions, and simulated microgravity caused a burst of ROS, implicating ROS as a critical upstream mediator linking to downstream atrophic signaling. ROS generation activated the ERK1/2 early-growth response protein (Egr)1/2-Cbl-b signaling pathway, an established contributing pathway to muscle volume loss. Interestingly, antioxidant treatments such as N-acetylcysteine and TEMPOL, but not catalase, blocked the clinorotation-mediated activation of ERK1/2. The increased ROS induced transcriptional activity of Egr1 and/or Egr2 to stimulate Cbl-b expression through the ERK1/2 pathway in L6 myoblasts, since treatment with Egr1/2 siRNA and an ERK1/2 inhibitor significantly suppressed clinorotation-induced Cbl-b and Egr expression, respectively. Promoter and gel mobility shift assays revealed that Cbl-b was upregulated via an Egr consensus oxidative responsive element at -110 to -60 bp of the Cbl-b promoter. Together, this indicates that under microgravity conditions, elevated ROS may be a crucial mechanotransducer in skeletal muscle cells, regulating muscle mass through Cbl-b expression activated by the ERK-Egr signaling pathway.

Rats deficient C-type natriuretic peptide suffer from impaired skeletal growth without early death.

We have previously investigated the physiological role of C-type natriuretic peptide (CNP) on endochondral bone growth, mainly with mutant mouse models deficient in CNP, and reported that CNP is indispensable for physiological endochondral bone growth in mice. However, the survival rate of CNP knockout (KO) mice fell to as low as about 70% until 10 weeks after birth, and we could not sufficiently analyze the phenotype at the adult stage. Herein, we generated CNP KO rats by using zinc-finger nuclease-mediated genome editing technology. We established two lines of mutant rats completely deficient in CNP (CNP KO rats) that exhibited a phenotype identical to that observed in mice deficient in CNP, namely, a short stature with severely impaired endochondral bone growth. Histological analysis revealed that the width of the growth plate, especially that of the hypertrophic chondrocyte layer, was markedly lower and the proliferation of growth plate chondrocytes tended to be reduced in CNP KO rats. Notably, CNP KO rats did not have malocclusions and survived for over one year after birth. At 33 weeks of age, CNP KO rats persisted significantly shorter than wild-type rats, with closed growth plates of the femur in all samples, which were not observed in wild-type rats. Histologically, CNP deficiency affected only bones among all body tissues studied. Thus, CNP KO rats survive over one year, and exhibit a deficit in endochondral bone growth and growth retardation throughout life.

Measurements of ionic concentrations along with endocochlear potential in wild-type and claudin 14 knockout mice.

OBJECTIVE: To examine whether the changes in endolymphatic ion concentrations were involved in hair cells degeneration in claudin-14 knockout (KO) mice (Cldn14-/-), we measured the endocochlear potential (EP) along with concentrations of K+, Na+, H+, or Ca2+ ([K]e, [Na]e, pHe, [Ca]e) in Cldn14-/-, in which hair cells were selectively damaged, and compared with measurements in wild type mice (Wt). METHODS: We used the Cldn14-/- from 3 weeks of age, in which the auditory brain responses (ABR) was severely diminished. Using double-barreled ion-selective microelectrodes, we measured [K]e, [Na]e, pHe, and [Ca]e in both Wt and Cldn14-/- at 8-10 weeks of age. RESULTS: (1) In Wt, the EP was +92mV. [K]e, [Na]e, pHe, and [Ca]e were 169mM, ∼1.0mM, 7.50, and 395nM, respectively. In the Cldn14-/-, the EP was +96mV. [K]e, [Na]e, pHe, and [Ca]e were 167mM, ∼1.0mM, 7.73, and 179nM, respectively. No significant differences in the above values were observed between Wt and Cldn14-/-. (2) A significant linear correlation between EP and [Ca]e (R=0.93) was observed for both Wt and Cldn14-/-, but no correlation was observed between EP and K+, Na+, or H+. CONCLUSION: These findings suggest that (1) the changes in endolymphatic ion concentrations might not be involved in hair cells degeneration in Cldn14-/-, (2) [Ca]e might be regulated by EP in both Wt and Cldn14-/-.

Effects of growth hormone on thyroid function are mediated by type 2 iodothyronine deiodinase in humans.

PURPOSE: Growth hormone (GH) therapy in adults alters thyroid function, and acromegaly often involves thyroid disease. The present study aimed to elucidate roles and mechanisms of GH in regulating thyroid function. METHODS: We performed two retrospective observational studies, which focused on consecutive patients with severe adult GH deficiency who received recombinant human GH (rhGH) therapy (n = 20) and consecutive patients with acromegaly who underwent transsphenoidal surgery (TSS) (n = 25). In both studies, serum free triiodothyronine (fT3), free thyroxine (fT4), and fT3/fT4 ratio were examined before and after the interventions. We subsequently administered GH to four human cell lines (HepG2, TSA201, MCF7, and HTC/C3) in vitro, and examined changes in mRNA levels of iodothyronine deiodinases (D1, D2, and D3). RESULTS: Median serum fT3 level significantly increased after rhGH therapy from 2.38 to 2.78 pg/mL (p < 0.001), and fT4 decreased from 1.115 to 1.065 ng/dL (p = 0.081). TSS significantly decreased median serum fT3 from 3.03 to 2.53 pg/mL (p < 0.001), and increased fT4 from 1.230 to 1.370 ng/dL (p < 0.001). In vitro, GH significantly increased D2 expression at the mRNA level in HTC/C3 cells (p < 0.01), as well as D2 protein and its activity. CONCLUSIONS: GH increased serum fT3 level and decreased serum fT4 level in humans. Our results suggest that its mechanism involves D2 upregulation. Considering this GH effect on thyroid hormone metabolism, data on thyroid function could be useful in the management of GH deficiency and acromegaly.