Satoshi Matsuyama

Selection of autophagy or apoptosis in cells exposed to ER-stress depends on ATF4 expression pattern with or without CHOP expression

Summary Cells exposed to ER-stress undergo the Unfolded Protein Response (UPR) to avoid apoptosis, but may also activate autophagy. However, the signal for selection of one of these two protective responses is unknown. To clarify the key switch between autophagy and apoptosis, we examined the correlation of UPR-related signals with autophagy and/or apoptosis inductions in HepG2 cells exposed to three ER-stress inducers (NaF, tunicamycin, and thapsigargin) with time, including the effect of small interfering RNA on the cell responses. Thapsigargin-induced ER-stress caused only apoptosis after ∼2 hr with Ire1 phosphorylation, and Grp78, ATF4, and CHOP expressions. On the other hand, NaF- and tunicamycin-induced ER-stress caused only autophagy in the early stage by ∼8 hr with ATF4 expression and without CHOP expression. ATF4-siRNA completely inhibited the autophagy induced by NaF or tunicamycin with suppressed ATF4 protein and mRNA expressions, and also inhibited apoptosis by thapsigargin with suppression of both ATF4 and CHOP. CHOP-siRNA had no effect on autophagy activation by NaF and tunicamycin. On the other hand, CHOP-siRNA activated autophagy in thapsigargin-induced ER-stress with significant ATF4 expression, and suppressed apoptosis with CHOP suppression. These results showed that ATF4 is the key signal for autophagy induced by ER-stress, and that autophagy is switched to apoptosis by subsequent CHOP upregulation, suggesting that the changeover switch between autophagy and apoptosis is located between ATF4 to CHOP in the PERK pathway.

Enhancement of estrogen receptor gene expression in the mediobasal hypothalamus during anestrus in the beagle bitch.

Estrogen receptor mRNA (ER mRNA) levels were measured in the mediobasal hypothalamus (MBH) of beagle bitches at different stages of the estrous cycle, and compared with levels in ovariectomized (OVX) estrogen-treated bitches. In cyclic bitches, the level of hypothalamic ER mRNA increased during the progression of anestrus and declined thereafter. Hypothalamic ER mRNA and plasma luteinizing hormone (LH) levels during anestrus and proestrus were positively correlated (r = 0.94, P < 0.001). In OVX bitches, levels of hypothalamic ER mRNA were low, and increased significantly after treatment with a low dose of estradiol benzoate. These results suggest that, during the course of anestrus in the bitch, hypothalamic ER mRNA expression increases, and may be up-regulated by estradiol.

Increased lh pulse frequency and estrogen secretion associated with termination of anestrus followed by enhancement of uterine estrogen receptor gene expression in the beagle bitch

The relationships among pulsatile LH secretion pattern, estrogen secretion, and expression of the uterine estrogen receptor gene were examined throughout the estrous cycle in beagle bitches. In Experiment 1, blood samples were collected from 30 bitches every 10 min for 8 h from a cephalic vein during different phases of the estrous cycle. An increase in the mean plasma levels of LH occurred from mid to late anestrus (P < 0.01). The LH pulse frequency increased (P < 0.01) from late anestrus to proestrus, and was strongly correlated (r = 0.96, P < 0.001) with the mean plasma level of estradiol-17 beta (E2). In Experiment 2, middle uterine samples, including the myometrium and endometrium, from 18 bitches were taken at 6 stages of the estrous cycle. The total number of estrogen receptors and nuclear estrogen receptor and its mRNA levels in the uterus also increased (P < 0.01) from late anestrus to proestrus. Mean plasma E2 level and the number of uterine estrogen receptor were positively correlated (r = 0.81, P < 0.05). In Experiment 3, nine bitches were ovariectomized in mid anestrus. Two weeks later they received a single injection of 10 or 50 micrograms/kg, i.m., estradiol benzoate. The number of uterine estrogen receptor and their mRNA levels for ovariectomized bitches were low, but increased (P < 0.05) after treatment with a low dose of estradiol benzoate. These results suggest that increases in LH pulse frequency and estrogen secretion are associated with termination of anestrus and that subsequent enhancement of uterine estrogen receptor expression may be up-regulated by estradiol.

Enhancement of aromatase gene expression in the mediobasal hypothalamus during anestrus in the beagle bitch.

The relationships among expression of cytochrome p450 aromatase (p450arom) mRNA in the mediobasal hypothalamus (MBH), ovarian aromatase activity, and estrogen secretion were examined throughout the estrous cycle in beagle bitches. Using polymerase chain reaction (PCR) analysis we were able to detect p450arom gene transcripts in the canine MBH. The level of hypothalamic p450arom mRNA increased during the progression of anestrus and declined thereafter. Ovarian p450arom activity, as measured by a (3)H2O assay, were low in anestrus, increased in proestrus, and declined thereafter. Ovarian p450arom activity and plasma estradiol-17beta levels were positively correlated (r=0.77, P<0.05). These results suggest that enhancement of hypothalamic p450arom gene expression is associated with termination of anestrus.

Detection of transforming growth factor-α and epidermal growth factor receptor mRNA and immunohistochemical localization of their proteins in the ovine uterus during the early implantation period

Accumulated evidence suggests that growth factors of the epidermal growth factor (EGF) family play an important role in the murine implantation process. In the sheep, however, the uterine distribution of these factors and their receptor, EGF receptor (EGF-R), during implantation is not known. This study examined the presence of mRNA transcripts and immunohistochemical localization for transforming growth factor-α (TGF-α), the potent EGF-family member, and EGF-R in the ovine uterus during the early implantation period. By reverse transcriptase-polymerase chain reaction and sequencing of the products, the presence of TGF-α and EGF-R mRNA transcripts were detected in the endometrium on Days 14, 16 and 20 (Day 0 = day of mating). Immunohistochemical analysis revealed that the luminal and glandular epithelial cells and some stromal cells of the endometrium and the trophectoderm were positive for TGF-α and EGF-R on Days 14 and 15. Distinct staining for TGF-α was observed in the glandular epithelium of deep endometrial areas and strong immunoreactivity for EGF-R was found in the trophectoderm. On Days 16, 18 and 20, although the staining pattern for TGF-α was similar to that on the previous days, the immunoreactivity for EGF-R in the stromal cells increased and that in the gland decreased. A distinct immunoreactivity for EGF-R was found in the trophectoderm throughout the days examined. These results suggest that TGF-α expressed in the endometrium and trophectoderm may exert effects locally on these tissues during implantation in sheep. Furthermore, it is speculated that the temporal changes in the uterine EGF-R distribution may be related to the endometrial microvascular development.

Expression and localization of epidermal growth factor, transforming growth factor-α and epidermal growth factor receptor in the canine testis

Gene expression of epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and EGF receptor (EGF-R) and the localization of the corresponding proteins in the canine testis were studied. Levels of mRNA expressions were determined by semiquantitative reverse transcription polymerase chain reaction in the testes of the peripubertal (4–6 months), young adult (3–4 years), advanced adult (7–8 years) and senescent (11–16 years) groups. The EGF-R mRNA level in the testes of the peripubertal group was significantly higher than those in the other groups, whereas there was no difference in EGF and TGF-α mRNA levels among groups. Immunohistochemical stainings for EGF, TGF-α and EGF-R in the testis revealed that immunoreactivity in the seminiferous epithelium and Sertoli cell was weak and nonspecific for the stage of spermatogenesis, and distinct staining was found in Leydig cells. These results suggest that the EGF family of growth factors may be involved in testicular maturation and function in the dog.

Hypersensitivity of mouse NEIL1-knockdown cells to hydrogen peroxide during S phase

Oxidative base damage occurs spontaneously due to reactive oxygen species generated as byproducts of respiration and other pathological processes in mammalian cells. Many oxidized bases are mutagenic and/or toxic, and most are repaired through the base excision repair pathway. Human endonuclease VIII-like protein 1 (hNEIL1) is thought to play an important role during the S phase of the cell cycle by removing oxidized bases in DNA replication fork-like (bubble) structures, and the protein level of hNEIL1 is increased in S phase. Compared with hNEIL1, there is relatively little information on the properties of the mouse ortholog mNEIL1. Since mouse cell nuclei lack endonuclease III-like protein (NTH) activity, in contrast to human cell nuclei, mNEIL1 is a major DNA glycosylase for repair of oxidized pyrimidines in mouse nuclei. In this study, we made mNEIL1-knockdown cells using an shRNA expression vector and examined the cell cycle-related variation in hydrogen peroxide (H2O2) sensitivity. Hypersensitivity to H2O2 caused by mNEIL1 knockdown was more significant in S phase than in G1 phase, suggesting that mNEIL1 has an important role during S phase, similarly to hNEIL1.

Abundance of BER-related proteins depends on cell proliferation status and the presence of DNA polymerase β.

In mammalian cells, murine N-methylpurine DNA glycosylase (MPG) removes bases damaged spontaneously or by chemical agents through the process called base excision repair (BER). In this study, we investigated the influence of POL β deficiency on MPG-initiated BER efficiency and the expression levels of BER-related proteins in log-phase and growth-arrested (G0) mouse embryonic fibroblasts (MEFs). G0 wild-type (WT) or POL β–deficient (Pol β–KO) cells showed greater resistance to methyl methanesulfonate than did log-phase cells, and repair of methylated bases was less efficient in the G0 cells. Apex1 mRNA expression was significantly lower in Pol β–KO or G0 WT MEFs than in log-phase WT MEFs. Moreover, although Mpg mRNA levels did not differ significantly among cell types, MPG protein levels were significantly higher in log-phase WT cells than in log-phase Pol β–KO cells or either type of G0 cells. Additionally, proliferating cell nuclear antigen protein levels were also reduced in log-phase Pol β–KO cells or either type of G0 cells. These results indicated that MPG-initiated BER functions mainly in proliferating cells, but less so in G0 cells, and that POL β may be involved in regulation of the amount of intracellular repair proteins.

Immunohistochemical analyses of the kinetics and distribution of macrophages in the developing rat kidney

Macrophages are required during kidney development and appear in the initiation and propagation of renal injury. To establish baseline data, we analyzed the kinetics of the macrophage with different immunophenotypes in the developing rat kidney (fetus at 18 and 20 days, neonate at 1-21 days, and adult at 7-weeks old). Macrophages reacting to CD68, CD163, and MHC class II were identified in the cortex and medulla of the developing rat kidney. CD68+ macrophages appeared in the fetal kidney as early as fetal day 18, and the number increased gradually in the neonatal kidney, whereas MHC class II+ and CD163+ macrophages first appeared on neonatal days 4 and 8, respectively. Apoptotic bodies were seen in the fetal kidney and early stages of the neonatal kidney (days 1–4), and simultaneously CD68+ macrophages appeared, indicating that CD68+ macrophages may have roles in phagocytosis of apoptotic bodies and contribute to renal tissue maturation. Colony stimulating factor 1 and insulin growth factor 1 mRNAs were increased in the late stage of renal development (neonatal day 12 or later), and simultaneously CD163+ and MHC class II+ cells appeared, suggesting that these cells may be a source of these growth factors and participate in renal tissue modeling. Generally, the CD163+ and MHC class II+ cell number was much smaller than that of CD68+ cells in the developing neonatal kidney. Therefore, the obtained findings provide valuable information on the participation of macrophages in the developing rat kidney. This information may be useful for evaluation of renal toxicity when macrophages are involved in the development of renal injury.

Cloning and expression analysis of prohibitin mRNA in canine mammary tumors

Prohibitin is an antiproliferative protein that is a product of a putative tumor suppressor gene. However, there is little information on prohibitins in companion animals. In this study, we cloned canine prohibitin mRNA using RT-PCR and 3′-RACE (Rapid Amplification of cDNA Ends). The sequence was well conserved compared with those of other mammals, including human. The deduced amino acid sequence translated from the open reading frame completely corresponded to the human sequence. Canine prohibitin mRNA was expressed in all normal mammary and tumor samples examined. These results suggest that this protein plays a vital role in cell growth mechanisms and may be related to the occurrence of canine mammary tumors.