Growth Factor Messenger RNA Levels Research Articles

Myostatin, follistatin and activin type II receptors are highly expressed in adenomyosis

To evaluate the expression pattern of activins and related growth factor messenger RNA (mRNA) levels in adenomyotic nodules and in their endometrium. Prospective study. University hospital. Symptomatic premenopausal women scheduled to undergo hysterectomy for adenomyosis. Samples from adenomyotic nodules and homologous endometria were collected. Endometrial tissue was also obtained from a control group. Quantitative real-time polymerase chain reaction (PCR) analysis and immunohistochemical localization of activin-related growth factors (activin A, activin B, and myostatin), binding protein (follistatin), antagonists (inhibin-α, cripto), and receptors (ActRIIa, ActRIIb) were performed. Myostatin mRNA levels in adenomyotic nodule were higher than in eutopic endometrium and myostatin, activin A, and follistatin concentrations were higher than in control endometrium. No difference was observed for inhibin-α, activin B, and criptomRNA levels. Increased mRNA levels of ActRIIa and ActRIIb were observed in adenomyotic nodules compared with eutopic endometrium and control endometrium. Immunofluorescent staining for myostatin and follistatin confirmed higher protein expression in both glands and stroma of patients with adenomyosis than in controls. The present study showed for the first time that adenomyotic tissues express high levels of myostatin, follistatin, and activin A (growth factors involved in proliferation, apoptosis, and angiogenesis). Increased expression of their receptors supports the hypothesis of a possible local effect of these growth factors in adenomyosis. The augmented expression of ActRIIa, ActRIIb, and follistatin in the endometrium of these patients may play a role in adenomyosis-related infertility.

NcRuPAR inhibits gastric cancer progression by down-regulating protease-activated receptor-1.

ncRuPAR is a newly discovered long noncoding RNA molecule that can upregulate protease-activated receptor-1 (PAR-1) during embryonic growth; however, its role in cancer has not been elucidated. Here, we conducted a study to investigate the role of ncRuPAR in gastric cancer. Significant downregulation of ncRuPAR was detected in gastric cancer tissues compared with paired adjacent nontumor tissues; however, both PAR-1 and vascular endothelial growth factor (VEGF) messenger RNA (mRNA) levels were significantly higher in cancerous tissues compared with adjacent normal tissues. Additionally, the expression level of ncRuPAR was found to be significantly correlated with tumor invasion depth, lymph node metastasis, distant metastasis, tumor size, and tumor-nodes-metastasis (TNM) stage and inversely associated with the mRNA levels and extent (E) × intensity (I) scores of PAR-1 and VEGF. The protein level of PAR-1 was significantly correlated with tumor size only, while the VEGF protein level was significantly correlated with invasion depth and tumor size. The area under the receiver operating characteristic (ROC) curve of ncRuPAR was 0.84 (95 % CI 0.79-0.88) at a cutoff value of 4.97; ncRuPAR had a sensitivity of 88.41 %, a specificity of 73.91 %, and an accuracy of 81.16 % for the prediction of gastric cancer. These results suggest that ncRuPAR inhibits gastric cancer development, and its underlying mechanism involves the inhibition of PAR-1. In addition, ncRuPAR could be regarded as a marker for gastric cancer in the future.

Effects of L-carnitine on fetal growth and the IGF system in pigs1,2

The effects of L-carnitine on porcine fetal growth traits and the IGF system were determined. Fourth-parity sows were fed a gestation diet with either a 50-g top dress containing 0 (control, n = 6) or 100 mg of L-carnitine (n = 6). At midgestation, fetuses were removed for growth measurements, and porcine embryonic myoblasts (PEM) were isolated from semitendinosus. Real-time quantitative PCR was used to measure growth factor messenger RNA (mRNA) levels in the uterus, placenta, muscle, hepatic tissue, and cultured PEM. A treatment x day interaction (P = 0.02) was observed for maternal circulating total carnitine. Sows fed L-carnitine had a greater (P = 0.01) concentration of total carnitine at d 57 than control sows. Circulating IGF-I was not affected (P = 0.55) by treatment. Supplementing sows with L-carnitine resulted in larger (P = 0.02) litters (15.5 vs. 10.8 fetuses) without affecting litter weight (P = 0.07; 1,449.6 vs. 989.4 g) or individual fetal weight (P = 0.88) compared with controls. No treatment effect was found for muscle IGF-I (P = 0.36), IGF-II (P = 0.51), IGFBP-3 (P = 0.70), or IGFBP-5 (P = 0.51) mRNA abundance. The abundance of IGF-I (P = 0.72), IGF-II (P = 0.34), and IGFBP-3 (P = 0.99) in hepatic tissue was not influenced by treatment. Uterine IGF-I (P = 0.46), IGF-II (P = 0.40), IGFBP-3 (P = 0.29), and IGFBP-5 (P = 0.35) mRNA abundance did not differ between treatments. Placental IGF-I (P = 0.30), IGF-II (P = 0.18), IGFBP-3 (P = 0.94), and IGFBP-5 (P = 0.42) mRNA abundance did not differ between treatments. There was an effect of side of the uterus for IGF-I (P = 0.04) and IGF-II (P = 0.007) mRNA abundance; IGF-I mRNA abundance was greater in the left uterine horn than in the right uterine horn (0.14 and 0.07 relative units, respectively). Placental IGF-II mRNA abundance was greater (P = 0.007) in the left than in the right uterine horn (483.5 and 219.59, respectively). The abundance of IGFBP-3 was not affected by uterine horns in either uterine (P = 0.66) or placental (P = 0.13) tissue. There was no treatment difference for IGF-I (P = 0.31) or IGFBP-5 (P = 0.13) in PEM. The PEM isolated from sows fed L-carnitine had decreased IGF-II (P = 0.02), IGFBP-3 (P = 0.03), and myogenin (P = 0.04; 61, 59, and 67%, respectively) mRNA abundance compared with controls. These data suggest that L-carnitine supplemented to gestating sows altered the IGF system and may affect fetal growth and development.

Growth factor messenger RNA levels in muscle and liver of steroid-implanted and nonimplanted steers.

Ribonuclease protection assays were used to measure steady-state semimembranosus muscle and/or hepatic levels of IGF-I, IGFBP-3, IGFBP-5, hepatocyte growth factor (HGF), and myostatin messenger RNA (mRNA) in steers implanted from 32 to 38 d with Revalor-S, a combined trenbolone acetate and estradiol implant. Insulin-like growth factor-ImRNA levels were 69% higher (P < 0.01, n = 7) in the livers of implanted steers than in the livers of nonimplanted steers. Similarly, IGF-I mRNA levels were 50% higher (P < 0.05, n = 7) in the semimembranosus muscles of implanted steers than in the same muscles from nonimplanted steers. Hepatic IGFBP-3 mRNA levels were 24% higher (P < 0.07, n = 7) in implanted steers than in nonimplanted steers. Hepatic HGF and IGFBP-5 mRNA levels did not differ between implanted and nonimplanted steers. Similarly, muscle IGFBP-3, IGFBP-5, HGF, and myostatin mRNA levels were not affected by treatment. Previous data from these same steers have shown that circulating IGF-I and IGFBP-3 concentrations were 30 to 40% higher (P < 0.01, n = 7) in implanted steers than in nonimplanted, control steers. Additionally, the number of actively proliferating satellite cells that could be isolated from the semimembranosus muscle was 45% higher (P < 0.01, n = 7) for implanted steers than for nonimplanted steers. Viewed together, these data suggest that increased muscle IGF-I levels stimulate increased satellite cell proliferation, resulting in the increased muscle growth observed in Revalor-S implanted steers.

Regulation of amniotic fluid volume by intramembranous absorption in sheep:: Role of passive permeability and vascular endothelial growth factor

Objective: During long-term intravascular fluid infusion in the ovine fetus, a large increase in fetal urinary flow rate occurs while amniotic fluid volume increases only slightly because of increased intramembranous absorption. The current study tested the hypotheses that passive intramembranous permeability increases in response to fetal intravascular saline solution infusion and that the increased intramembranous absorption occurs in parallel with an increase in vascular endothelial growth factor gene expression in the amnion, chorion, and placenta. Study Design: Chronically catheterized fetal sheep that average 126 ± 1 (SE) days of gestation either were infused intravascularly with 7 L of normal saline solution over 3 days (n = 8 sheep) or served as time controls (n = 6 sheep). Amniotic fluid volume and fetal urinary flow rate were measured daily. Intramembranous diffusional permeability was estimated daily as being equal to the clearance of intra-amniotically injected technetium 99m. Vascular endothelial growth factor messenger RNA abundance in the amnion, chorion, and placenta was determined by Northern blot analysis. Statistical analyses included analysis of variance. Results: In the infused fetuses, amniotic fluid volume and urinary flow increased (P <.01) by 891 ± 144 mL and 3488 ± 487 mL per day, respectively, on infusion day 3 compared with no changes over time in the control fetuses. In the infused fetuses, estimated intramembranous absorption increased by 4276 ± 499 mL during the 3-day infusion. Intramembranous technetium 99m permeability was similar over time in the two groups. In the infused group, vascular endothelial growth factor messenger RNA levels in the amnion, chorion, and placenta increased 2- to 4-fold compared with the control group (P <.001). Conclusion: The up-regulation of vascular endothelial growth gene expression may mediate the increase in the intramembranous absorption that is induced by volume-loading diuresis; however, this does not occur by passive mechanisms. We speculate that vascular endothelial growth mediates the increased intramembranous absorption by increasing vesicular transport. (Am J Obstet Gynecol 2003;188:786-93.)

Prolonged hypoxia upregulates vascular endothelial growth factor messenger RNA expression in ovine fetal membranes and placenta

Objective: In ovine fetuses, 4 days of hypoxia resulted in a large increase in urine flow, without the development of polyhydramnios, which suggests that intramembranous absorption of the amniotic fluid was enhanced. Because vascular endothelial growth factor is speculated to be a regulator of intramembranous absorption through increases of membrane vascularity and fluid transport, we hypothesized that hypoxia upregulated vascular endothelial growth factor gene expression in the fetal membranes. Study Design: Five near-term ovine fetuses that were subjected to 4 days of hypoxia and 5 age-matched time controls were studied. On day 4, the amnion, chorion, and placenta were collected for cellular localization and quantification of vascular endothelial growth factor messenger RNA and for the determination of vascular endothelial growth factor molecular forms that were expressed. The data were analyzed statistically with the use of t tests and 2-factor analyses of variance. Results: Vascular endothelial growth factor messenger RNA was expressed in the fetal membranes localized to the amniotic epithelium and chorionic cytotrophoblast, and to the villous cytotrophoblast of the placenta. In hypoxic fetuses, vascular endothelial growth factor messenger RNA levels in these cell layers were significantly increased compared with the controls. Five vascular endothelial growth factor molecular forms were identified with vascular endothelial growth factor164 being the most abundant form expressed. The pattern of expression of the forms was not altered by hypoxia. Conclusion: In the near-term ovine fetus, hypoxia induced vascular endothelial growth factor messenger RNA expression in the amnion, chorion, and placenta. This was associated with an increase in intramembranous absorption of amniotic fluid. We speculate that the increased intramembranous absorption was mediated by a vascular endothelial growth factor-induced increase in the transport of amniotic fluid into the fetal membranes. (Am J Obstet Gynecol 2002;186:303-10.)

Differential patterns of nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 mRNA and protein levels in developing regions of rat brain.

The present studies were undertaken to characterize the regional and temporal patterns of neurotrophin messenger RNA and protein levels for beta-nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in the developing CNS. We have examined the levels of these neurotrophin messenger RNAs with ribonuclease protection assays and corresponding protein levels with enzyme-linked immunosorbent assays in the developing Long-Evans rat hippocampus, neocortex and cerebellum on postnatal days 1, 7, 14, 21, and 92. In addition, immunohistochemistry was used to localize the neurotrophins in these developing brain regions. Results indicated that in neocortex and hippocampus, messenger RNA for both nerve growth factor and brain-derived neurotrophic factor increased in an age-dependent manner, reaching a plateau by postnatal day 14. In the neocortex, nerve growth factor and brain-derived neurotrophic factor protein levels both peaked at postnatal day 14. In hippocampus, nerve growth factor protein peaked at postnatal day 7 while brain-derived neurotrophic factor peaked at postnatal day 14. In cerebellum, nerve growth factor messenger RNA levels were flat, while nerve growth factor protein peaked at postnatal day 7. Brain-derived neurotrophic factor messenger RNA increased in an age-dependent manner while the pattern for its protein levels was mixed. Neurotrophin-3 messeger RNA levels increased in an age-dependent manner in hippocampus, peaked at postnatal day14 in cerebellum, and no changes occurred in neocortex. Neurotrophin-3 protein was at its peak at postnatal day 1 and thereafter decreased at other postnatal days in all three brain regions. Results of neurotrophin immunohistochemistry often paralleled and complemented enzyme-linked immunosorbent assay data, demonstrating specific cell groups containing neurotrophin proteins in these regions. Within each region, patterns with regard to messenger RNA and respective protein levels for each neurotrophin were unique. No consistent relationship between patterns of neurotrophin messenger RNAs and their cognate proteins was observed between regions. The different regional patterns for neurotrophin messengerRNA and protein levels in each brain region indicate that messenger RNA studies of neurotrophin messenger RNA must be augmented by protein determination to fully characterize spatial and temporal neurotrophin distribution.

Hypoxia activates jun-N-terminal kinase, extracellular signal-regulated protein kinase, and p38 kinase in pulmonary arteries.

Chronic alveolar hypoxia is the major cause of pulmonary hypertension. The cellular mechanisms involved in hypoxia- induced pulmonary arterial remodeling are still poorly understood. Mitogen-activated protein kinase (MAPK) is a key enzyme in the signaling pathway leading to cellular growth and proliferation. The purpose of this investigation was to determine the roles that MAPKs, specifically Jun-N-terminal kinase (JNK), extracellular signal-regulated protein kinase (ERK), and p38 kinase, play in the hypoxia-induced pulmonary arterial remodeling. Rats were exposed to normobaric hypoxia (10% O(2)) for 1, 3, 7, or 14 d. Hypoxia caused significant remodeling in the pulmonary artery characterized by thickening of pulmonary arterial wall and increases in tissue mass and total RNA. JNK, ERK, and p38 kinase tyrosine phosphorylations and their activities were significantly increased by hypoxia. JNK activation peaked at Day 1 and ERK/p38 kinase activation peaked after 7 d of hypoxia. The results from immunohistochemistry show that hypoxia increased phospho-MAPK staining in both large and small intrapulmonary arteries. Hypoxia also upregulated vascular endothelial growth factor messenger RNA (mRNA) and platelet-derived growth factor receptor mRNA levels in pulmonary artery with a time course correlated to the activation of ERK and p38 kinase. The gene expressions of c-jun, c-fos, and egr-1, known as downstream effectors of MAPK, were also investigated. Hypoxia upregulated egr-1 mRNA but downregulated c-jun and c-fos mRNAs. These data suggest that hypoxia-induced activation of JNK is an early response to hypoxic stress and that activation of ERK and p38 kinase appears to be associated with hypoxia-induced pulmonary arterial remodeling.

Hepatocyte growth factor induces rat ovarian surface epithelial cell mitosis or apoptosis depending on the presence or absence of an extracellular matrix.

The present studies showed that sequential treatment with equine CG (eCG) and hCG not only induced an increase in ovarian weight, but also caused an estimated 4.6-fold increase in the number of ovarian surface epithelial cells. In addition, eCG-hCG treatment increased ovarian hepatocyte growth factor (HGF) messenger RNA levels. These studies also demonstrated that rat primary ovarian surface epithelial cells as well as a cell line derived from rat ovarian surface epithelium (i.e. ROSE-179 cells) do not express the LH (hCG) receptor. Both of these cells express c-Met, the receptor for HGF. To assess the effects of hCG and HGF on ovarian surface epithelial cell mitosis, ROSE-179 cells were cultured for 24 h in serum-supplemented medium on either glass or the synthetic fibronectin-like extracellular matrix protein, pronectin (RGD). The cells were then cultured for 24 h in serum-free medium in the presence or absence of hCG or HGF. The numbers of cells at 2, 24, and 48 h of culture were determined. The percentage of apoptotic cells was assessed by in situ DNA staining at 48 h of culture. In the serum-supplemented medium in the presence or absence of RGD, the number of ROSE-179 cells doubled. In serum-free medium, cell proliferation was reduced, and the percentage of apoptotic nuclei ranged between 10-15% regardless of the substrate. Neither mitosis nor apoptosis was influenced by hCG in the presence or absence of RGD. For ROSE-179 cells cultured in serum-free medium on RGD, HGF induced mitosis, resulting in a 2.8 +/- 0.2-fold increase in cell number compared with the 24 h control values. On a glass substrate in serum-free medium, HGF did not induce mitosis, but increased the percentage of apoptotic nuclei. Time-lapse photographic analysis revealed that on RGD, cells undergoing HGF-induced mitosis showed a transient reduction in cell contact. On glass, HGF caused many cells to completely lose contact and separate from each other. Collectively, these data suggest that in vivo gonadotropins stimulate HGF expression and ovarian surface epithelial cell proliferation. Based on in vitro studies, it is likely that the mitogenic action of hCG is mediated by HGF. However, HGF only induces mitosis in the presence of an extracellular matrix.

Vascular endothelial growth factor is more important than basic fibroblastic growth factor during ischemic wound healing.

To test the influence of vascular endothelial growth factor (VEGF) on normal and ischemic wounds in a noncontractive dermal ulcer standardized model in the rabbit ear and to assay the levels of both VEGF and basic fibroblastic growth factor messenger RNA levels in normal and ischemic wounds at different intervals during the healing process. Dermal ulcers were created in the normal and ischemic ears of 20 anesthetized young female New Zealand white rabbits. Either VEGF 121, VEGF 165 (30 microg per wound), or buffered saline solution alone was applied to each wound and covered. Wounds were harvested at day 7 or 10 and evaluated histologically. Twenty-four similar rabbits were wounded in the same manner and their untreated wounds were harvested at 1, 3, 7, and 10 days after wounding. The wounds were analyzed with reverse transcriptase polymerase chain reaction. Histologic specimens were measured for amount of new epithelium and granulation tissue. Reverse transcriptase polymerase chain reaction was used to determine basic fibroblastic growth factor and VEGF messenger RNA expression. Both isoforms of VEGF improved granulation tissue formation in both normal and ischemic wounds with a magnitude similar to other vulnerary agents tested in the past. Vascular endothelial growth factor application had no effect on new epithelium formation. In contrast to basic fibroblastic growth factor, VEGF messenger RNA levels were induced 4 fold by ischemia alone and 6 fold by wounding in both ischemic and normal wounds. Vascular endothelial growth factor seems to be more important than basic fibroblastic growth factor during ischemic wound healing. Treatment of ischemic wounds with VEGF improves the deficit in wound healing produced by ischemia.

Spatial learning and physical activity contribute to the induction of fibroblast growth factor: neural substrates for increased cognition associated with exercise

New evidence indicates that neural activity regulates the expression of trophic factors in the brain but regulation of these molecules by select aspects of behaviour remains solely a fascinating possibility. We report that following training in the Morris water maze, a spatial memory task, the hippocampus and cerebellum of learning rats exhibited an increase in basic fibroblast growth factor messenger RNA. Basic fibroblast growth factor messenger RNA levels were higher during the learning of the task and decreased once asymptotic performance was reached, suggesting an involvement of basic fibroblast growth factor in learning/memory. An active control group, which exercised for the same time as the learning group but the spatial learning component of the task was minimized, exhibited a minor increase in basic fibroblast growth factor messenger RNA. The intensification of the physical activity component of the task by massed or intensive training resulted in greater increases in basic fibroblast growth factor messenger RNA for both learning and yoked groups, but levels of basic fibroblast growth factor messenger RNA in the learning group remained higher than yoked only in the cerebellum. Changes in basic fibroblast growth factor were accompanied by an increase in astrocyte density in the hippocampus in agreement with described roles of basic fibroblast growth factor in astrocyte proliferation/reactivity. Results suggest that learning potentiates the effects of physical activity on trophic factor induction in select brain regions. Trophic factor involvement in behaviour may provide a molecular basis for the enhanced cognitive function associated with active lifestyles, and guide development of strategies to improve rehabilitation and successful ageing.

Ontogeny of epidermal growth factor (EGF), EGF receptor (EGFR) and basic fibroblast growth factor (bFGF) mRNA levels in pancreas, liver, kidney, and skeletal muscle of pig

Epidermal growth factor (EGF), EGF receptor (EGFR), and basic fibroblast growth factor (bFGF) messenger RNA (mRNA) levels were examined by Northern blot analysis in four tissues (pancreas, liver, kidney, and skeletal muscle) of pig from fetal 90 d to postnatal 180 d of age. The present study shows for the first time that EGF mRNA increased with advancing age in the kidney and skeletal muscle of pig. A high level of EGF mRNA was observed in the kidney compared with the liver and skeletal muscle. In the pancreas, high levels of EGF mRNA were found in fetuses and newborns and were low in older pigs. Pancreatic EGFR mRNA level parallelled its EGF mRNA, whereas in the kidney and skeletal muscle, patterns of EGFR mRNA were reversed to their EGF mRNA levels. In the liver, EGFR mRNA was abundant but EGF mRNA was undetected. In the pancreas and skeletal muscle, the highest levels of bFGF mRNA were found in fetuses of 90 d of age and then decreased with advancing age. In the liver and kidney, there were no major changes in bFGF mRNA levels during the examined developmental periods. These results show that EGF, EGFR, and bFGF mRNA levels are developmentally and tissue specifically regulated in pig. In the pancreas, mRNA levels of EGF, EGFR and bFGF were high in fetal and neonatal life and low thereafter. In the kidney and skeletal muscle, EGF mRNA increased with advancing age. EGF may play a role in muscle growth and maintenance in growing pigs during the later stage of development.

Effect of weaning on epidermal growth factor and its receptor messenger RNA levels in various tissues of piglets

Weights of pancreas, liver, and stomach, and total DNA of pancreas and stomach mucosa per body weight were greater (P &lt; 0.05) in weaned piglets than suckling ones at 30 d of age. There was no effect (P &gt; 0.05) of weaning on epidermal growth factor (EGF) and EGF receptor (EGFR) messenger RNA levels in liver, stomach, duodenum, and skeletal muscle. Messenger RNAs of EGF in pancreas increased following weaning in piglets of 27 (P = 0.0001) and 30 (P = 0.004) d of age, indicating a possible involvement of EGF in pancreatic development during this period. Key words: Weaning, EGF, EGFR mRNA, Pig tissues

Chronic continuous infusion of (−)nicotine reduces basic fibroblast growth factor messenger RNA levels in the ventral midbrain of the intact but not of the 6-hydroxydopamine-lesioned rat

A negative correlation has been found between smoking and Parkinson's disease. There is evidence to suggest that this correlation appears to be associated with a neuroprotective role of nicotine for dopamine neurons at least in relation to mechanical injury. However, 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP) neurotoxicity to dopamine neurons is enhanced by chronic continuous (-)nicotine. More recently, basic fibroblast growth factor has been found to possess neurotrophic activities for many nerve cells including the dopamine cells in vivo and in vitro. Therefore, it is of interest to explore a possible effect of nicotine on basic fibroblast growth factor expression in the ventral midbrain of intact and 6-hydroxydopamine-lesioned rats and how treatment with nicotine can alter the 6-hydroxydopamine-induced injury of the nigral dopamine nerve cells as evaluated by dopamine biochemistry. In the present paper, an analysis of the effects of chronic continuous infusion of (-)nicotine via minipumps was carried out on basic fibroblast growth factor expression in the vental midbrain of the intact male rat and of the 6-hydroxydopamine lesioned rat. A quantitative messenger RNA protection assay analysis was used as well as an immunocytochemical analysis in the substantia nigra. Our findings give evidence that a two-week continuous infusion with (-)nicotine in the intact rat leads to substantial and dose-related (0.03-0.3 mg/kg per h) reductions of basic fibroblast growth factor messenger RNA levels in the ventral midbrain. These changes are not associated with changes in neuronal and glial basic fibroblast growth factor immunoreactivity in this region with the antibodies used. However, a one-week continuous infusion with (-)nicotine (0.125 mg/kg per h) failed to significantly alter the basic fibroblast growth factor messenger RNA levels in the ventral midbrain of solvent and 6-hydroxydopamine-injected rats and thus also the 6-hydroxydopamine-induced increase of basic fibroblast growth factor messenger RNA levels in the ventral midbrain of the lesioned side observed at this time-interval and known to be of astroglial origin [Chadi G. et al. (1994) Neuroscience 61, 891-910]. In agreement, the 6-hydroxydopamine-induced depletion of dopamine in the neostriatum was unaltered by the nicotine treatment (0.125 mg/kg per h). Thus, chronic continuous (-)nicotine treatment may lead to a reduced basic fibroblast growth factor trophic tone in the ventral midbrain of the intact but not of the 6-hydroxydopamine-lesioned rat neither on the lesioned nor on the unlesioned side of the ventral midbrain. It seems possible that chronic nicotine treatment mainly reduces basic fibroblast growth factor messenger RNA levels of neuronal origin, since the astroglial messenger RNA levels dominate after the 6-hydroxydopamine-induced lesions.

Cell-specific modulation of basal and seizure-induced neurotrophin expression by adrenalectomy.

Reports of glucocorticoid effects on neurotrophin expression suggest that adrenal hormones may contribute to the pattern of changes in the expression of these factors induced by neuronal activity and seizures. To examine this possibility, the present study evaluated the influence of adrenalectomy on basal expression and seizure-induced alterations in levels of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 messenger RNAs in hippocampus, entorhinal cortex, and superficial neocortex. For determination of hormone effects on basal expression, adult male rats were adrenalectomized and killed 10-14 days later with paired adrenal-intact controls. For studies of adrenal steroid involvement in expression following seizure, adrenalectomized and adrenal-intact rats received a seizure-producing lesion of the dentate gyrus hilus. Changes in neurotrophin messenger RNA content were assessed by quantitative in situ hybridization. Adrenalectomy alone had no significant effect on brain-derived neurotrophic factor messenger RNA content but did result in cell-specific decreases in nerve growth factor and neurotrophin-3 messenger RNAs. Nerve growth factor messenger RNA levels were reduced in hippocampal stratum granulosum, entorhinal cortex, and neocortex but not in cells of the hippocampal molecular layers or hilus. With adrenalectomy, neurotrophin-3 messenger RNA was virtually eliminated from CA2 stratum pyramidale, partially reduced in stratum granulosum, but unaffected in neurons of the hippocampal molecular layers or entorhinal cortex. These effects were partially reversed by corticosterone (2 mg/l) supplement to the drinking saline. In experimental-seizure rats, adrenalectomy did not alter the direction or basic pattern of seizure-induced changes in neurotrophin expression but did change the time courses and magnitudes of these effects. In all areas measured, brain-derived neurotrophic factor messenger RNA content was more greatly and persistently elevated by seizure in adrenalectomized as compared with adrenal-intact rats. In contrast, with adrenalectomy seizures induced smaller increases in nerve growth factor messenger RNA content. Adrenalectomy augmented the decrease in neurotrophin-3 messenger RNA induced by seizure in hippocampus but not in entorhinal cortex. These results demonstrate that adrenal hormones play a major role in the regulation of basal nerve growth factor and neurotrophin-3 messenger RNA expression by specific populations of forebrain neurons. Moreover, the adrenal steroids have opposite effects on activity-dependent changes in brain-derived neurotrophic factor and nerve growth factor messenger RNA levels but are not required for the basic pattern of changes in neurotrophin messenger RNA expression elicited by recurrent seizures.

Changes in nerve growth factor and preprotachykinin messenger RNA levels in the iris and trigeminal ganglion in diabetic rats; effects of treatment with insulin or nerve growth factor

This study was designed to explore effects of experimental diabetes mellitus on expression of substance P in the trigeminal ganglion and of nerve growth factor (NGF) in the iris. Rats with streptozotocin-diabetes showed an increase in NGF mRNA in the iris (P < 0.01) which was corrected by insulin treatment. There was also an increase in the levels of mRNA for the substance P precursor, preprotachykinin (PPT), in the trigeminal ganglion of these animals, despite there being no change in GAP-43 mRNA. Since expression of both of these peptides is sensitive to NGF in vitro, we examined the effect of treatment of diabetic rats with NGF at three different doses (0.2, 0.5 and 1.0 mg/kg body weight). There was a dose-dependent increase in both gamma-PPT and GAP-43 mRNA in the trigeminal ganglia of NGF-treated diabetic rats. The findings indicate that increased NGF may be responsible for raised substance P levels in the iris.

Effect of in utero ethanol exposure on the postnatal ontogeny of insulin-like growth factor-1, and type-1 and type-2 insulin-like growth factor receptors in the rat brain

There is convincing evidence that alcohol consumption during pregnancy causes major CNS abnormalities; however, the molecular and cellular basis of these dysfunctions is currently not understood. This study examined the effects of prenatal ethanol exposure on the expression of insulin-like growth factor-1 messenger RNA and type-1 and type-2 receptor protein and messenger RNA expression in the developing rat brain. Mothers were maintained on an ethanol containing liquid diet from day 2 of pregnancy through parturition and the offspring were killed at birth, 10, 20 and 40 days of age. Insulin-like growth factor-1 messenger RNA, and insulin-like growth factor receptors demonstrated developmentally dependent expression in specific brain regions throughout the postnatal period of CNS maturation. Insulin-like growth factor-1 gene expression in the brain, as analysed by dot-blot hybridization, was greatest at birth, and decreased 61% in ad libitum and pair-fed animals by 20 days of age. In contrast, ethanol-treated animals exhibited only a 25% decrease in insulin-like growth factor-1 messenger RNA levels during the same period. This delay in insulin-like growth factor-1 messenger RNA maturation may be related to a developmental delay in CNS development in the prenatally ethanol exposed offspring. Prenatal ethanol exposure did not alter the observed localization of insulin-like growth factor-1 messenger RNA. While alterations were observed in long-term insulin-like growth factor-1 messenger RNA regulation, quantitative receptor autoradiography and in situ hybridization demonstrated no alterations in either type-1 or type-2 insulin-like growth factor receptor populations in ethanol-treated animals. Changes in hepatic and plasma insulin-like growth factor-1 and insulin-like growth factor-binding protein regulation have also been observed in these animals, suggesting changes in protein translation and the autocrine/paracrine actions of this peptide. The present study demonstrated that insulin-like growth factor-1 messenger RNA and insulin-like growth factor receptors are regionally expressed during early postnatal development and that ethanol administration influenced the long-term regulation of insulin-like growth factor messenger RNA levels in the brain without affecting either its localization or insulin-like growth factor receptor populations. These results, in combination with previous results from our laboratory on the effects of prenatal ethanol exposure on plasma insulin-like growth factor-1 and insulin-like growth factor-binding proteins, provide additional evidence that even modest levels of prenatal ethanol exposure enhance the possibility of alterations in growth promoting peptides in the fetus and neonate, leading to an increased risk of fetal alcohol effects in the offspring.

Deficits in sciatic nerve neuropeptide content coincide with a reduction in target tissue nerve growth factor messenger RNA in streptozotocin-diabetic rats: Effects of insulin treatment

The role of sub-optimal neurotrophic support in the aetiology of the sensory neuron dysfunction associated with diabetic neuropathy was investigated. The status of sciatic nerve neuropeptide content was related to target tissue nerve growth factor messenger RNA levels in streptozotocin-diabetic rats. The levels of substance P and calcitonin gene-related peptide in diabetic sciatic nerve were significantly lowered by approximately 50% and 28%, respectively, compared with aged matched controls and insulin-treated diabetic rats ( P < 0.01) for both peptides and both comparisons). Measurements of nerve growth factor messenger RNA levels in sensory neuron target tissues, namely foot-skin and soleus muscle, revealed deficits of approximately 50% in diabetic rats, with insulin treatment reversing the decrease in foot-skin but not in soleus muscle. The results show a possible correlation between deficient neuropeptide gene expression in sensory neurons and reduced nerve growth factor messenger RNA levels in target tissue.

Increased gene expression of scatter factor—hepatocyte growth factor and basic fibroblast growth factor in granulation tissue in the rat

Scatter factor-hepatocyte growth factor is a protein secreted by fibroblasts which disperses colonies of epithelial cells and keratinocytes in culture. The factor is also a patent mitogen for hepatocytes, synthesized in the liver. Basic fibroblast growth factor, another heparin-binding factor, is most abundant in the brain but also plays a role in wound healing. Using a solution hybridization/RNAase protection assay, we have measured the abundance of messenger RNA for scatter factor-hepatocyte growth factor and basic fibroblast growth factor in granulation tissue obtained from subcutaneously Hunt-Schilling wound cylinders. The levels of scatter factor-hepatocyte growth factor messenger RNA increased after weeks 2 through 4 to a twofold higher level in weeks 5 through 7 after implantation of the cylinders, whereas no changes in basic fibroblast growth factor messenger RNA levels were noticed. At week 3 after implantation of the cylinders, scatter factor-hepatocyte growth factor messenger RNA levels in granulation tissue were more than threefold higher than in skin dermis fibroblasts but markedly lower than in the liver. The abundance of basic fibroblast growth factor messenger RNA was also significantly increased in granulation tissue compared with dermis but, as expected, markedly lower than in the brain. In conclusion, the gene expression of the scatter factor-hepatocyte growth factor, as well as basic fibroblast growth factor, is increased in granulation tissue. Because there was a time-dependent increase in the expression of scatter factor-hepatocyte growth factor, it is hypothesized that scatter factor-hepatocyte growth factor acts as a signal from fully developed granulation tissue to stimulate skin epithelial cells to scatter over the wound.

Activation of protein kinase-C differentially regulates insulin-like growth factor-I and basic fibroblast growth factor messenger RNA levels

Activation of protein kinase-C differentially regulates insulin-like growth factor-I and basic fibroblast growth factor messenger RNA levels