Biomedical subjects

D Chrysis

Publications and source records attributed to D Chrysis.

8 recordsLinked to original sources

PubMed2006

Locally produced estrogen promotes fetal rat metatarsal bone growth; an effect mediated through increased chondrocyte proliferation and decreased apoptosis.

The importance of estrogens for the regulation of longitudinal bone growth is unequivocal. However, any local effect of estrogens in growth plate cartilage has been debated. Recently, several enzymes essential for estrogen synthesis were shown to be expressed in rat growth plate chondrocytes. Local production of 17beta-estradiol (E2) has also been demonstrated in rat costal chondrocytes. We aimed to determine the functional role of locally produced estrogen in growth plate cartilage. The human chondrocyte-like cell line HCS-2/8 was used to study estrogen effects on cell proliferation (3H-labeled thymidine uptake) and apoptosis (cell death detection ELISA kit). Chondrocyte production of E2 was measured by RIA and organ cultures of fetal rat metatarsal bones were used to study the effects of estrogen on longitudinal growth rate. We found that significant amounts of E2 were produced by HCS-2/8 chondrocytes (64.1 +/- 5.3 fmol/3 days/10(6) cells). The aromatase inhibitor letrozole (1 microM) and the pure estrogen receptor antagonist ICI 182,780 (10 microM) inhibited proliferation of HCS-2/8 chondrocytes by 20% (P < 0.01) and almost 50% (P < 0.001), respectively. Treatment with ICI 182,780 (10 microM) increased apoptosis by 228% (P < 0.05). Co-treatment with either caspase-3 or pan-caspase inhibitors completely blocked ICI 182,780-induced apoptosis (P < 0.001 vs ICI 182,780 only). Moreover, both ICI 182,780 (10 microM) and letrozole (1 microM) decreased longitudinal growth of fetal rat metatarsal bones after 7 days of culture (P < 0.01). In conclusion, our data clearly show that chondrocytes endogenously produce E2 and that locally produced estrogen stimulates chondrocyte proliferation and protects from spontaneous apoptosis. In addition, longitudinal growth is promoted by estrogens locally produced within the epiphyseal growth plate.

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PubMed2003

Growth retardation induced by dexamethasone is associated with increased apoptosis of the growth plate chondrocytes.

Glucocorticoids cause significant growth retardation in mammals and humans and decreased proliferation of chondrocytes has been considered as the main local mechanism. Death by apoptosis is an important regulator of homeostasis in multicellular organisms. Here we chose to study the role of apoptosis in growth retardation caused by glucocorticoid treatment. We treated 7-week-old male rats with dexamethasone (5 mg/kg/day) for 7 days. Apoptosis was studied in tibiae growth plates by the TUNEL method. Immunoreactivity for parathyroid hormone-related peptide (PTHrP), caspase-3, and the anti-apoptotic proteins Bcl-2 and Bcl-x was also studied. Apoptosis was mainly localized in terminal hypertropic chondrocytes (THCs) in both control and dexamethasone-treated animals. Dexamethasone caused an increase in apoptosis which was fourfold in THCs (2.45+/-0.12 vs 0.62+/-0.09 apoptotic cells/mm growth plate, P<0.001), and 18-fold in proliferative chondrocytes (0.18+/-0.04 vs 0.01+/-0.007 apoptotic cells/mm growth plate, P<0.001). Increased apoptosis after dexamethasone treatment was accompanied by increased immunoreactivity for caspase-3 and decreased immunoreactivity for the anti-apoptotic proteins Bcl-2 and Bcl-x, which further supports our apoptosis results. Dexamethasone also decreased the immunoreactivity for PTHrP, suggesting a role in the mechanism by which glucocorticoids induce apoptosis in the growth plate. We conclude that apoptosis is one mechanism involved in growth retardation induced by glucocorticoids. Premature loss of resting/proliferative chondrocytes by apoptosis could contribute to incomplete catch-up seen after prolonged glucocorticoid treatment.

Animals↗

PubMed2002

Estrogen receptor-alpha and -beta are expressed throughout postnatal development in the rat and rabbit growth plate.

Estrogen regulates skeletal growth and promotes epiphyseal fusion. To explore the mechanisms underlying these effects we investigated the expression of estrogen receptor-alpha (ERalpha) and -beta (ERbeta) in rat and rabbit growth plates during postnatal development, using immunohistochemistry. Immunoreactivity for ERalpha and ERbeta was observed in resting zone and proliferative zone chondrocytes at all ages studied for both rat (7, 14, 28 and 70 days of age) and rabbit (1, 7, 28 and 120 days of age). In the rat distal humerus and the rabbit proximal tibia, expression of both receptors in the hypertrophic zone was minimal at early ages, increasing only at the last time point prior to epiphyseal fusion. Expression was rarely seen in the hypertrophic zone of the rat proximal tibia, a growth plate that does not fuse until late in life. Therefore, we conclude that ERalpha and ERbeta are both expressed in the mammalian growth plate. The temporal and anatomical pattern suggests that ER expression in the hypertrophic zone in particular may play a role in epiphyseal fusion.

Aging↗

PubMed2001

Insulin-like growth factor-I overexpression attenuates cerebellar apoptosis by altering the expression of Bcl family proteins in a developmentally specific manner.

In studies of transgenic (Tg) mice that overexpress insulin-like growth factor-I (IGF-I) exclusively in the CNS, we demonstrated a dramatic increase in cerebellar granule cell number that appeared to be attributable predominantly to enhanced survival. IGF-I anti-apoptotic actions are well established in cultured neurons, but comparable studies in vivo are few. Using the same Tg mice, therefore, we set out to document IGF-I anti-apoptotic effects during cerebellar development and to probe IGF-I signaling mechanisms. Compared with cerebella (CBs) of non-Tg littermates, those of Tg mice had fewer apoptotic cells at postnatal day 7 (P7) and showed a similar tendency at P14 and P21. At each age studied, procaspase-3 and caspase-3 were decreased in CBs of Tg mice. The caspase-3 decline was accompanied by decreases in the 85 kDa fragment of Poly(ADP-ribose) polymerase, a known product of caspase cleavage, suggesting decreased caspase activity. At P7 decreased apoptosis in Tg mice was associated with increased expression of the anti-apoptotic Bcl genes, Bcl-x(L) and Bcl-2. The mRNA expression of the proapoptotic Bcl genes, Bax and Bad, also was increased, but no changes were observed in the abundance of their proteins. At P14 Bcl-xL and Bcl-2 expression were similar in normal and Tg mice; Bax mRNA was unchanged in Tg mice, but its protein abundance was decreased, and both Bad mRNA and protein abundance were decreased. At P21 Bcl-xL and Bcl-2 expression were unchanged, but Bax and Bad expression were decreased. Our data show that IGF-I exerts anti-apoptotic actions during cerebellar development, and thereby alters the magnitude of naturally occurring apoptosis. IGF-I appears to affect multiple steps in the apoptotic pathway in a developmentally specific manner. IGF-I decreases caspase-3 availability and activity, increases the expression of anti-apoptotic Bcl-x(L) and Bcl-2 during early postnatal development, and decreases proapoptotic Bax and Bad expression at later developmental stages.

Aging↗

PubMed1999

Regulation of components of the ubiquitin system by insulin-like growth factor I and growth hormone in skeletal muscle of rats made catabolic with dexamethasone.

To investigate whether the anabolic effects of insulin-like growth factor I (IGF-I) and GH are mediated through regulation of the ubiquitin (Ub) pathway, we examined the effect of IGF-I (0.35 microg/100 g) and/or GH (0.3 mg/100 g BW) on the expression of Ub and Ub-conjugating (E2) enzyme messenger RNAs (mRNAs) in skeletal muscle of rats made catabolic by treatment with dexamethasone (Dex; 0.5 mg/100 g BW) for 3 days. Dex caused a significant loss of body and gastrocnemius weight (14% and 25%, respectively) concurrent with an increase in the 2.8- and 1.2-kb transcripts of Ub (14.3- and 12-fold increases, respectively), the 1.8- and 1.2-kb transcripts of 14-kDa Ub-conjugating enzyme (E2-14 kDa; 5.6- and 7.7-fold increases, respectively), the 4.4- and 2.4-kb transcripts of Ub-E2G (6.5- and 8.2-fold increases, respectively), and the 2E isoform of the 17-kDa E2 mRNA (3.5-fold increase). Injections of IGF-I in Dex-treated animals ameliorated the body weight loss, and the gastrocnemius tended to be heavier. This improvement was also accompanied by a significant suppression of transcripts for Ub (58% and 66% decreases), E2-14 kDa (58% and 68% decreases), and Ub-E2G (78% decrease), whereas the 2E isoform of the 17-kDa E2 was only modestly affected (20% decrease). GH restored serum IGF-I levels to normal in Dex-treated rats, but had no effect on the body weight loss or on any of the studied components of the Ub pathway. Administration of IGF-I to the Dex/GH-treated animals decreased the activated mRNAs of the Ub pathway in a manner and degree similar to those observed in the Dex/ IGF-I group. In summary, these results provide evidence that IGF-I regulates the expression of mRNAs encoding components of the Ub pathway during catabolism and suggest a possible mechanism for the antiproteolytic actions of IGF-I. On the other hand, GH, which is believed not to affect proteolysis but only protein synthesis, had no effect on any of the mRNAs studied.

Animals↗

PubMed1998

Reduction of hepatic insulin-like growth factor I (IGF-I) messenger ribonucleic acid (mRNA) during fasting is associated with diminished splicing of IGF-I pre-mRNA and decreased stability of cytoplasmic IGF-I mRNA.

The mechanisms by which fasting decreases liver insulin-like growth factor I (IGF-I) messenger RNA (mRNA) abundance have not been defined completely. In the present study, we have examined the effects of fasting in rats on hepatic IGF-I gene transcription, IGF-I pre-mRNA splicing, and cytoplasmic IGF-I mRNA stability. Using the in vitro nuclear run-on transcription technique, we observed that fasting did not change IGF-I gene transcription activity [76 +/- 32 densitometric units (DU) for fasted vs. 58 +/- 23 DU for control-fed rats; P = 0.1], whereas IGF-binding protein-1 (IGFBP-1) gene transcription, a positive control, was increased more than 2-fold (729 +/- 157 DU for fasted vs. 261 +/- 56 DU for control-fed rats; P < 0.05). This implies that fasting-induced reduction of liver IGF-I mRNA is due to events other than a decreased rate of IGF-I gene transcription. By measuring nonspliced (pre-mRNA) and spliced IGF-I transcripts in liver nuclear RNA using ribonuclease protection assays, we found that IGF-I pre-mRNA was increased in fasted rats (measured as the percentage of beta-actin: 34.0 +/- 5.5% for fasted vs. 8.1 +/- 3.8% for control-fed rats; P < 0.01), whereas spliced IGF-I transcript remained unchanged (measured as the percentage of beta-actin: 60.9 +/- 9.2% for fasted vs. 79.0 +/- 6.2% for control-fed rats; P = 0.75). We then compared this pattern of splicing to IGF-I pre-mRNA splicing in hypophysectomized rats subjected to GH stimulation and to IGFBP-1 pre-mRNA splicing in the same fasting experiment. One hour after GH injection, we observed a coordinate increase in both nonspliced and spliced IGF-I transcripts in liver nuclei of hypophysectomized rats. Fasting increased both IGFBP-1 pre-mRNA and spliced transcript. Taken together, these results indicate that the increase in IGF-I pre-mRNA in liver nuclei during fasting is caused by delayed pre-mRNA splicing, rather than increased IGF-I gene transcription. To examine the possible effect of fasting on hepatic IGF-I mRNA stability, we used an in vitro model of nutrient deprivation (fewer amino acids in culture medium) of rat hepatocyte primary culture. Each of the three major IGF-I mRNA species exhibited a shortened half-life in the amino acid-deprived media. The 7.5-kb IGF-I mRNA, however, was degraded faster than the two smaller IGF-I mRNA species. This may indicate that fasting decreases the stability of liver IGF-I mRNA in vivo. In summary, these results suggest that fasting regulates hepatic IGF-I gene expression mainly at the posttranscriptional level by delaying IGF-I pre-mRNA splicing, which attenuates mature IGF-I mRNA generation, and by accelerating the rate of degradation of IGF-I mRNA in cytoplasm.

Actins↗

PubMed1998

Effect of fasting on insulin receptor-related receptor messenger ribonucleic acid in rat kidney.

The insulin receptor-related receptor (IRR), a member of the insulin receptor tyrosine kinase family, has structural homology to the insulin receptor (IR) and the IGF-I receptor (IGF-IR). The ligand, gene regulation and biological function of the IRR are not known. Because mRNAs for both the IR and IGF-IR are increased by nutrient restriction, we used RNase protection assays to assess the effects of fasting 48 h on IRR mRNA in kidneys of rats. We compared the changes in IRR with those in IR and IGF-IR mRNAs. We observed a significant increase in steady state levels of IRR (ratio of IRR mRNA to beta-actin in fed P<0.01), suggesting that the ligand for IRR also might be regulated by nutrients.

Animals↗

PubMedDate not supplied

Programmed cell death in normal fetal rat lung development.

Lung development is a coordinated process regulated by the interactions of extracellular and intracellular factors, yet little is known about the process of programmed cell death during lung development. To study this question, we examined fetal rat lung from the pseudoglandular period (gestational day 15) to the day of birth (gestational day 21) using BrdU incorporation into DNA as a proliferative marker, while in parallel examining several markers of programmed cell death including terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), DNA "laddering, " and expression of programmed cell death pathway proteins. Cell proliferation was ongoing throughout fetal days 15 to 21 with a decrease in proliferation over days 20 and 21. Programmed cell death in fetal lung also appeared to be present at all ages examined, but demonstrated 2 peaks of activity at fetal days 15 and 18 to 20. Bcl-XL expression was detected on fetal days 15 to 21, with diminished expression on days E15 to E18. Cleaved poly(ADP-ribose)polymerase (PARP), activated caspase-3, Bax, and Bad were increased on days 18 to 20. We conclude that proliferation is the primary process driving fetal lung development with programmed cell death occurring throughout the lung developmental process to refine structural remodeling.

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