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During enamel formation, transforming growth factor-beta (TGF-β) isoforms exhibit different activities for gene expression, apoptosis, and endocytosis. This study aimed to investigate the differential response of TGF-β isoforms to epithelial-mesenchymal transition (EMT) in enamel epithelial cells.Using a mouse enamel epithelial cell line (mHAT9d) cultured in the presence of each TGF-β isoform, (1) the morphological changes in EMT were explored, (2) EMT-related genes were analyzed by next-generation sequencing (NGS), (3) TGF-β pathway for EMT was identified by inhibition experiments, and (4) the expression of the TGF-β receptor gene in response to the binding affinity of the TGF-β isoform were analyzed.EMT was observed in mHAT9d cultured in the presence of TGF-β1 and β3 but not TGF-β2. The expression of both epithelial and mesenchymal marker genes was observed in mHAT9d exhibiting EMT. NGS analysis suggested extracellular signal-regulated kinase (ERK) and Rho pathways as TGF-β signaling pathways associated with EMT. However, EMT in mHAT9d cultured in the presence of TGF-β1 or β3 occurred even in presence of an ERK1/2 inhibitor and was suppressed by Rho-kinase inhibitor. The expression of co-receptors for TGF-β signaling in mHAT9d cells reduced following stimulation with each TGF-β isoform. In contrast, endoglin levels increased following TGF-β1 or β3 stimulation, but no change was noted in response to TGF-β2.We propose that in TGF-β-stimulated enamel epithelial cells, EMT mainly occurred via the Rho signaling pathway, and the differences in response across TGF-β isoforms were due to their endoglin-mediated binding affinity for the TGF-β receptor.

Background In recent years, transforming growth factor (TGF)-β has been found in the enamel matrix, and along with enamel protein, is thought to play an important role in the process of calcification of tooth enamel. The purpose of this study was to investigate the dynamics of TGF-β and its interactions with enamel proteins, through experiments at the genetic and protein levels. Highlights The expression of the latent TGF-β1 gene was observed during the enamel formation process. TGF-β1 was found in both immature and mature enamel, and its activity tended to decrease as immature enamel transitioned to mature enamel. In vitro studies showed that latent TGF-β1 was activated by matrix metalloproteinase 20 (MMP20), and activated TGF-β1 was degraded by kallikrein-4 (KLK4). By binding to the major amelogenins, activated TGF-β1 maintained its activity. Of the major amelogenins, the 13 kDa amelogenin had the highest affinity for activated TGF-β1. Moreover, the 13 kDa amelogenin-activated TGF-β1 complex was bound to the TGF-β1 receptor on the ameloblast cell surface and induced signaling. Conclusion Latent TGF-β1 produced and secreted from secretory-stage ameloblasts is activated by MMP20, and the activated TGF-β1 maintains its activity by combining with amelogenin cleavage products processed by the same protease. TGF-β1 moves through the aqueous phase with the water-soluble 13 kDa amelogenin and binds to its receptor on the ameloblast surface, thereby inducing autocrine signaling. Once the ameloblasts differentiate and enter the maturation stage, TGF-β1 is degraded by KLK4, which is produced and secreted by maturation-stage ameloblasts, and loses its activity.

Objective To provide in vivo biochemical evidence for the isolation, identification, and characterization of porcine keratin 75 (K75) in developing enamel. Methods Immunolocalization of K75 was observed in mandibles from mice at postnatal days 5 and 11. K75 gene expression was analyzed by quantitative reverse transcription-polymerase chain reaction using enamel organ epithelium (EOE) of incisors from pigs at 5 months of age. Enamel protein was extracted and isolated from both immature and mature enamel of second molars from 5-month-old pigs, and the K75 antibody-positive fraction was analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). In vitro protease digestion of K75-antibody-positive fraction was carried out using porcine kallikrein 4 (pKLK4) or recombinant human enamelysin (rhMMP20) and their degradation patterns were characterized by both SDS-PAGE and western blotting. Results Specific immunostaining for K75 was restricted to the layers of stratum intermedium and the enamel side of ameloblasts in mice at postnatal day 5, and to the papillary layer at postnatal day 11. Porcine K75 was expressed throughout enamel formation, but its transcript levels were significantly higher in the transition EOE than in the secretory- and maturation-stage EOE. Porcine K75 was extracted from the neutral soluble fraction from both immature and mature enamel. It was identified by LC-MS/MS analysis, and was found not to be degraded by either pKLK4 or rhMMP20. Conclusion We propose that K75 is present in the developing enamel and undergoes different processing/degradation compared to other enamel proteins.

Objectives To investigate potential functions of transforming growth factor-beta (TGF-β) isoforms in maturation-stage ameloblasts during amelogenesis. Methods In vivo activation of TGF-β was characterized by using matrix metalloproteinase 20 null (Mmp20-/-) and wild-type (Mmp20+/+) mice. Using mHAT9d cells cultured in the presence of each TGF-β isoform, (1) cell proliferation was determined by MTS assay, (2) immunostaining with anti-cleaved caspase-3 monoclonal antibody was performed and apoptotic indices were measured, (3) gene expression was analyzed by RT-qPCR, and (4) the uptake of amelogenin into mHAT9d cells was directly observed using a fluorescence microscope. Results TGF-β1 and TGF-β3 were present in the enamel matrix of developing teeth which were activated by MMP20 in vivo. A genetic study revealed that the three TGF-β isoforms upregulate kallikrein 4 (KLK4) mRNA levels but downregulate carbonic anhydrase II. Moreover, TGF-β1 and TGF-β2 significantly upregulated the mRNA level of amelotin, whereas TGF-β3 dramatically downregulated the mRNA levels of odontogenic ameloblast-associated protein (ODAM), family with sequence similarity 83 member H (FAM83H), and alkaline phosphatase (ALP). Immunostaining analysis showed that the apoptosis of mHAT9d cells is induced by three TGF-β isoforms, with TGF-β3 being most effective. Both TGF-β1 and TGF-β3 induced endocytosis of amelogenin. Conclusions We propose that TGF-β is regulated in an isoform-specific manner to perform multiple biological functions such as gene expression related to the structure of basal lamina/ameloblasts, mineral ion transport, apoptosis, and endocytosis in maturation-stage ameloblasts.

Polarized epithelial cells exhibit a characteristic array of microtubules that are oriented along the apicobasal axis of the cells. The minus-ends of these microtubules face apically, and the plus-ends face toward the basal side. The mechanisms underlying this epithelial-specific microtubule assembly remain unresolved, however. Here, using mouse intestinal cells and human Caco-2 cells, we show that the microtubule minus-end binding protein CAMSAP3 (calmodulin-regulated-spectrin-associated protein 3) plays a pivotal role in orienting the apical-to-basal polarity of microtubules in epithelial cells. In these cells, CAMSAP3 accumulated at the apical cortices, and tethered the longitudinal microtubules to these sites. Camsap3 mutation or depletion resulted in a random orientation of these microtubules; concomitantly, the stereotypic positioning of the nucleus and Golgi apparatus was perturbed. In contrast, the integrity of the plasma membrane was hardly affected, although its structural stability was decreased. Further analysis revealed that the CC1 domain of CAMSAP3 is crucial for its apical localization, and that forced mislocalization of CAMSAP3 disturbs the epithelial architecture. These findings demonstrate that apically localized CAMSAP3 determines the proper orientation of microtubules, and in turn that of organelles, in mature mammalian epithelial cells.

Aims/Introduction Dipeptidyl peptidase-4 inhibitors and glinides are effective in reducing postprandial hyperglycemia. However, little information is available on the comparative effects of the two drugs on the levels of postprandial glucose. The aim of the present study was to compare the effects of sitagliptin and nateglinide on meal tolerance tests in drug-naive patients with type 2 diabetes mellitus. Materials and Methods The study participants were 19 patients with type 2 diabetes mellitus, which was inadequately controlled by diet and exercise. An open-label, prospective, cross-over trial was carried out to compare the effects of single-dose sitagliptin and nateglinide on the postprandial glucose level and its related hormones during meal tests. Results The change in area under the curve (AUC) of glucose from 0 to 180 min (AUC0–180 min) during the meal test by nateglinide was similar to that by sitagliptin. As expected, the change in active glucagon like peptide-1 was significantly higher after a single-dose of sitagliptin than nateglinide. Then, insulin secretion relative to glucose elevation (ISG) (ΔISG0–180 min: ΔAUC0–180 min insulin/AUC0–180 min glucose) was significantly enhanced by nateglinide compared with sitagliptin. Conversely, glucagon level (ΔAUC0–180 min glucagon) was increased by administration of nateglinide, whereas the glucagon level was reduced by administration of sitagliptin. Conclusions The effects of sitagliptin on postprandial glucose levels were similar to those of nateglinide in drug-naive type 2 diabetes patients. However, the induced changes in insulin, active glucagon-like peptide-1 and glucagon during meal loading suggest that reduction of postprandial hyperglycemia was achieved by the unique effect of each drug.

The present study was performed to examine how transforming growth factor β (TGF-β) in root-surrounding tissues on deciduous teeth regulates the differentiation induction into odontoclasts during physiological root resorption. We prepared root-surrounding tissues with (R) or without (N) physiological root resorption scraped off at three regions (R1–R3 or N1–N3) from the cervical area to the apical area of the tooth and measured both TGF-β and the tartrate-resistant acid phosphatase (TRAP) activities. The TGF-β activity level was increased in N1–N3, whereas the TRAP activity was increased in R2 and R3. In vitro experiments for the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation revealed that proteins from N1–N3 and R1–R3 enhanced the TRAP activity in RAW264 cells. A genetic study indicated that the mRNA levels of TGF-β1 in N1 and N2 were significantly increased, and corresponded with levels of osteoprotegerin (OPG). In contrast, the expression level of RANKL was increased in R2 and R3. Our findings suggest that TGF-β is closely related to the regulation of OPG induction and RANKL-mediated odontoclast differentiation depending on the timing of RANKL and OPG mRNA expression in the root-surrounding tissues of deciduous teeth during physiological root resorption.

Transforming growth factor-β1 (TGF-β1) is present in porcine enamel extracts and is critical for proper mineralization of tooth enamel. Here, we show that the mRNA of latent TGF-β1 is expressed throughout amelogenesis. Latent TGF-β1 is activated by matrix metalloproteinase 20 (MMP20), coinciding with amelogenin processing by the same proteinase. Activated TGF-β1 binds to the major amelogenin cleavage products, particularly the neutral-soluble P103 amelogenin, to maintain its activity. The P103 amelogenin-TGF-β1 complex binds to TGFBR1 to induce TGF-β1 signalling. The P103 amelogenin-TGF-β1 complex is slowly cleaved by kallikrein 4 (KLK4), which is secreted into the transition- and maturation-stage enamel matrix, thereby reducing TGF-β1 activity. To exert the multiple biological functions of TGF-β1 for amelogenesis, we propose that TGF-β1 is activated or inactivated by MMP20 or KLK4 and that the amelogenin cleavage product is necessary for the in-solution mobility of TGF-β1, which is necessary for binding to its receptor on ameloblasts and retention of its activity.

Patients with chronic heart failure (CHF) are frequently limited by muscle fatigue resulting from impaired skeletal muscle blood flow. Accordingly, we assessed working skeletal muscle oxygenation in such patients using near-infrared (NIR) spectroscopy. Nine normal subjects (mean age 52 years) and 12 patients with CHF (mean age 60 years) were studied. NIR spectroscopy was used to monitor relative changes in oxygenated hemoglobin (Hb) and myoglobin (Mb) (oxy Hb Mb ), deoxygenated Hb and Mb ( deoxy Hb Mb ), and total (oxy + deoxy) Hb and Mb (total Hb Mb ) contents in the vastus lateralis muscle at rest, during warm-up (0 W, 30 cycles/min to 3 min), incremental maximal supine bicycle exercise (ramp protocal, 15 W/min, 50 cycles/min), and recovery. At peak exercise the patients exhibited reduced heart rate, systolic blood pressure, peak exercise oxygen consumption (VO2; 15 ± 3.0 ml/kg.min vs 32 ± 8.5 ml/kg/min), workload (99 ± 23.4 W vs 183 ± 68.4 W) as compared with the normal subjects. The respiratory quotient was comparable in both groups. In the normal subjects, oxy Hb Mb was increased from the warm-up period to the early phase of exercise, followed by a progressive decrease to peak exercise. In the recovery phase, oxy Hb Mb was increased abruptly. For these patients, change in oxy Hb Mb followed a pattern similar to that seen in normal subjects, and oxy Hb Mb was decreased earlier in contrast to that in the normal subjects. There was a significant difference in the change of oxy Hb Mb during warm-up, early phase exercise, and recovery between the two groups. Changes in deoxy Hb Mb and oxy Hb Mb were inversely proportionate in both groups. In the recovery phase, the change in totla Hb Mb was smaller than that in the normal subjects. These data suggest that oxygen delivery to working skeletal muscle is impaired throughout exercise and a reactive hyperemic response to strenuous exercise is blunted in patients with CHF.

Background and Purpose Decreased exercise capacity is a major clinical problem in patients with chronic heart failure (CHF) and is associated with skeletal muscle fatigue and/or dyspnea. To study the factors contributing to the exercise limitation of CHF, we observed the oxygenation of both working skeletal and respiratory muscles during exercise. Method Thirteen subjects, 6 normal subjects and 7 patients with CHF, were studied. Using near-infrared (NIR) spectroscopy, relative changes in oxygenated Hb and Mb (oxy Hb/Mb) and deoxygenated Hb and Mb (deoxy Hb/Mb) contents in both vastus lateralis and serratus anterior muscles were monitored simultaneously during incremental maximal supine bicycle exercise (ramp protocol, 15 w/min, 50 cycle/min). Anaerobic threshold (AT) by respiratory gas analysis (VAT) was obtained using the V-slope method, and AT in the vastus lateralis (MAT) and the serratus anterior (RAT) muscles were determined by points obtained from deoxy Hb/Mb ≥ oxy Hb/Mb in each muscle. Results (1) In total subjects, VO2 at VAT was 10.1 ± 2.41 ml/min/kg (mean ± SD), VO2 at RAT 8.1 ± 1.80 and VO2 at MAT 7.0 ± 2.74 (VAT vs RAT p l 0.01, VAT vs MAT p l 0.01, RAT vs MAT P l 0.05). (2) In all of the subjects, there was significant correlation between VO2 at VAT and that at MAT (r = 0.608, p l 0.05), while there was no significant correlation between VO2 at VAT and that at RAT. The relations between VO2 at MAT and that at RAT are shown on the graph. Download : Download high-res image (85KB) Download : Download full-size image (3) Comparisons between patients with CHF and normal subjects. Download : Download high-res image (104KB) Download : Download full-size image Conclusions During exercise, the oxygenation profile of exercising skeletal muscle is similar to that of respiratory muscle, but the former shows anaerobic metabolism at an earlier stage of exercise than the latter. This phenomenon is more prominent in patients with CHF.