Enhanced Epidermal Growth Factor, Hepatocyte Growth Factor, and Vascular Endothelial Growth Factor Expression in Tuberous Sclerosis Complex
2011; Elsevier BV; Volume: 178; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2010.11.031
ISSN1525-2191
AutoresWhitney E. Parker, Ksenia Orlova, Gregory G. Heuer, Marianna Baybis, Eleonora Aronica, Michael Frost, Michael Wong, Peter B. Crino,
Tópico(s)Kruppel-like factors research
ResumoEpidermal growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) regulate angiogenesis and cell growth in the developing brain. EGF, HGF, and VEGF modulate the activity of the mammalian target of rapamycin (mTOR) cascade, a pathway regulating cell growth that is aberrantly activated in tuberous sclerosis complex (TSC). We hypothesized that expression of EGF, HGF, VEGF, and their receptors EGFR, c-Met, and Flt-1, respectively, would be altered in TSC. We show by cDNA array and immunohistochemical analysis that EGF, EGFR, HGF, c-Met, and VEGF, but not Flt-1, mRNA, and protein expression was up-regulated in Tsc1 conditional knockout (Tsc1GFAPCKO) mouse cortex. Importantly, these alterations closely predicted enhanced expression of these proteins in tuber and subependymal giant cell astrocytoma (SEGA) specimens in TSC. Expression of EGF, EGFR, HGF, c-Met, and VEGF protein, as well as hypoxia inducible factor-1α, a transcription factor that regulates VEGF levels and is also modulated by mTOR cascade activity, was enhanced in SEGAs (n = 6) and tubers (n = 10) from 15 TSC patients. Enhanced expression of these growth factors and growth factor receptors in human SEGAs and tubers and in the Tsc1GFAPCKO mouse may account for enhanced cellular growth and proliferation in tubers and SEGAs and provides potential target molecules for therapeutic development in TSC. Epidermal growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) regulate angiogenesis and cell growth in the developing brain. EGF, HGF, and VEGF modulate the activity of the mammalian target of rapamycin (mTOR) cascade, a pathway regulating cell growth that is aberrantly activated in tuberous sclerosis complex (TSC). We hypothesized that expression of EGF, HGF, VEGF, and their receptors EGFR, c-Met, and Flt-1, respectively, would be altered in TSC. We show by cDNA array and immunohistochemical analysis that EGF, EGFR, HGF, c-Met, and VEGF, but not Flt-1, mRNA, and protein expression was up-regulated in Tsc1 conditional knockout (Tsc1GFAPCKO) mouse cortex. Importantly, these alterations closely predicted enhanced expression of these proteins in tuber and subependymal giant cell astrocytoma (SEGA) specimens in TSC. Expression of EGF, EGFR, HGF, c-Met, and VEGF protein, as well as hypoxia inducible factor-1α, a transcription factor that regulates VEGF levels and is also modulated by mTOR cascade activity, was enhanced in SEGAs (n = 6) and tubers (n = 10) from 15 TSC patients. Enhanced expression of these growth factors and growth factor receptors in human SEGAs and tubers and in the Tsc1GFAPCKO mouse may account for enhanced cellular growth and proliferation in tubers and SEGAs and provides potential target molecules for therapeutic development in TSC. Tuberous sclerosis complex (TSC) is an autosomal dominant disorder that results from mutations in the TSC1 or TSC2 genes, which encode TSC1 and TSC2 proteins, respectively.1Consortium TECTSIdentification and characterization of the tuberous sclerosis gene on chromosome 16.Cell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1508) Google Scholar, 2van Slegtenhorst M. de Hoogt R. Hermans C. Nellist M. Janssen B. Verhoef S. Lindhout D. van den Ouweland A. Halley D. Young J. Burley M. Jeremiah S. Woodward K. Nahmias J. Fox M. Ekong R. Osborne J. Wolfe J. Povey S. Snell R.G. Cheadle J.P. Jones A.C. Tachataki M. Ravine D. Sampson J.R. Reeve M.P. Richardson P. Wilmer F. Munro C. Hawkins T.L. Sepp T. Ali J.B. Ward S. Green A.J. Yates J.R. Kwiatkowska J. Henske E.P. Short M.P. Haines J.H. Jozwiak S. Kwiatkowski D.J. Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.Science. 1997; 277: 805-808Crossref PubMed Scopus (1392) Google Scholar Many individuals with TSC exhibit cognitive disability and autism,3Bolton P.F. Park R.J. Higgins J.N. Griffiths P.D. Pickles A. Neuro-epileptic determinants of autism spectrum disorders in tuberous sclerosis complex.Brain. 2002; 125: 1247-1255Crossref PubMed Scopus (242) Google Scholar and more than 75% of TSC patients develop seizures.4Gomez M.R. Sampson J.R. Whittemore V.H. Tuberous Sclerosis Complex.in: Oxford University Press, New York1999: 340Google Scholar, 5Koh S. Jayakar P. Dunoyer C. Whiting S.E. Resnick T.J. Alvarez L.A. Morrison G. Ragheb J. Prats A. Dean P. Gilman J. Duchowny M.S. Epilepsy surgery in children with tuberous sclerosis complex: presurgical evaluation and outcome.Epilepsia. 2000; 41: 1206-1213Crossref PubMed Scopus (180) Google Scholar, 6Sparagana S.P. Roach E.S. Tuberous sclerosis complex.Curr Opin Neurol. 2000; 13: 115-119Crossref PubMed Scopus (113) Google Scholar Examination of the brain demonstrates cortical tubers and subependymal nodules (SENs) in more than 70% of TSC patients. Tubers are developmental malformations of the cerebral cortex highly associated with epilepsy and neurocognitive abnormalities. SENs are nodular lesions (typically smaller than 1 cm) located on the surfaces of the lateral and third ventricles. In approximately 10% to 20% of TSC patients, subependymal giant cell astrocytomas (SEGAs) arise within the lateral ventricles, often near the foramen of Monro. SEGAs are World Health Organization grade I tumors with low mitotic index as evidenced by Ki-67 immunoreactivity suggestive of slow cellular proliferation.7Sharma M. Ralte A. Arora R. Santosh V. Shankar S.K. Sarkar C. Subependymal giant cell astrocytoma: a clinicopathological study of 23 cases with special emphasis on proliferative markers and expression of p53 and retinoblastoma gene proteins.Pathology. 2004; 36: 139-144Crossref PubMed Scopus (36) Google Scholar, 8Gyure K.A. Prayson R.A. Subependymal giant cell astrocytoma: a clinicopathologic study with HMB45 and MIB-1 immunohistochemical analysis.Mod Pathol. 1997; 10: 313-317PubMed Google Scholar It is widely believed that SENs grow to form SEGAs, although the molecular mechanisms governing transformation from SEN to SEGA are unknown.9Kim S.K. Wang K.C. Cho B.K. Jung H.W. Lee Y.J. Chung Y.S. Lee J.Y. Park S.H. Kim Y.M. Choe G. Chi J.G. Biological behavior and tumorigenesis of subependymal giant cell astrocytomas.J Neurooncol. 2001; 52: 217-225Crossref PubMed Scopus (64) Google Scholar Both SENs and SEGAs consist of dysmorphic glial cells, enlarged giant cells (GCs), and spindle-shaped cells of unknown phenotype.10Lopes M.B. Altermatt H.J. Scheithauer B.W. Shepherd C.W. VandenBerg S.R. Immunohistochemical characterization of subependymal giant cell astrocytomas.Acta Neuropathol. 1996; 91: 368-375Crossref PubMed Scopus (89) Google Scholar, 11Mizuguchi M. Takashima S. Neuropathology of tuberous sclerosis.Brain Dev. 2001; 23: 508-515Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar Cellular immunoreactivity for glial fibrillary acidic protein (GFAP), neurofilament, S-100, neuron-specific enolase, and synaptophysin proteins suggests that SEGAs contain both glial and neuronal cell types. Lineage studies have demonstrated that SEGAs express cellular markers found in progenitors derived from the subventricular zone adjacent to the lateral ventricles12Lee A. Maldonado M. Baybis M. Walsh C.A. Scheithauer B. Yeung R. Parent J. Weiner H.L. Crino P.B. Markers of cellular proliferation are expressed in cortical tubers.Ann Neurol. 2003; 53: 668-673Crossref PubMed Scopus (70) Google Scholar, 13Ess K.C. Kamp C.A. Tu B.P. Gutmann D.H. Developmental origin of subependymal giant cell astrocytoma in tuberous sclerosis complex.Neurology. 2005; 64: 1446-1449Crossref PubMed Scopus (51) Google Scholar and that many of these markers are also expressed in cortical tubers. The TSC1 and TSC2 proteins combine to form a heterodimer that functions as an upstream modulator of the mammalian target of rapamycin (mTOR) pathway, which integrates growth factor and energy level signals to promote several cellular processes, including cell growth and proliferation,14Fingar D.C. Blenis J. Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression.Oncogene. 2004; 23: 3151-3171Crossref PubMed Scopus (1056) Google Scholar protein translation, and angiogenesis. TSC1 and TSC2 mutations are associated with loss of inhibitory modulation and consequent constitutive activation of the mTOR cascade, resulting in enhanced cell size and proliferation, especially under conditions favoring cell growth. Previous studies have suggested that altered growth factor expression may be associated with abnormal cellular architecture in the brains of TSC patients. For example, differential expression of neurotrophins and their receptors has been observed in cortical tubers,15Kyin R. Hua Y. Baybis M. Scheithauer B. Kolson D. Uhlmann E. Gutmann D. Crino P.B. Differential cellular expression of neurotrophins in cortical tubers of the tuberous sclerosis complex.Am J Pathol. 2001; 159: 1541-1554Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar and TSC1-TSC2 mediated control of mTOR is modulated by insulin-like growth factor-1 (IGF-1).16Harrington L.S. Findlay G.M. Gray A. Tolkacheva T. Wigfield S. Rebholz H. Barnett J. Leslie N.R. Cheng S. Shepherd P.R. Gout I. Downes C.P. Lamb R.F. The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins.J Cell Biol. 2004; 166: 213-223Crossref PubMed Scopus (925) Google Scholar, 17Shah O.J. Wang Z. Hunter T. Inappropriate activation of the TSC/Rheb/mTOR/S6K cassette induces IRS1/2 depletion, insulin resistance, and cell survival deficiencies.Curr Biol. 2004; 14: 1650-1656Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, 18Shah O.J. Hunter T. Turnover of the active fraction of IRS1 involves raptor-mTOR- and S6K1-dependent serine phosphorylation in cell culture models of tuberous sclerosis.Mol Cell Biol. 2006; 26: 6425-6434Crossref PubMed Scopus (135) Google Scholar Recent evidence also suggests that mTOR signaling is regulated by several growth factors, such as epidermal growth factor (EGF) and hepatocyte growth factor (HGF), and that TSC1-TSC2 may regulate downstream expression of select angiogenic factors, such as vascular endothelial growth factor (VEGF), via hypoxia inducible factor-1α (HIF-1α).19Brugarolas J. Kaelin Jr, W.G. Dysregulation of HIF and VEGF is a unifying feature of the familial hamartoma syndromes.Cancer Cell. 2004; 6: 7-10Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar For example, EGF regulates smooth muscle cell proliferation via its receptor EGFR through mTOR signaling.20Lesma E. Grande V. Ancona S. Carelli S. Di Giulio A.M. Gorio A. Anti-EGFR antibody efficiently and specifically inhibits human TSC2−/− smooth muscle cell proliferation: possible treatment options for TSC and LAM.PLoS One. 2008; 3: e3558Crossref PubMed Scopus (26) Google Scholar VEGF expression is up-regulated in the Eker rat TSC model, in mouse embryonic fibroblasts lacking Tsc2, and in facial angiofibromas from TSC patients.19Brugarolas J. Kaelin Jr, W.G. Dysregulation of HIF and VEGF is a unifying feature of the familial hamartoma syndromes.Cancer Cell. 2004; 6: 7-10Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 21Nguyen-Vu P.A. Fackler I. Rust A. DeClue J.E. Sander C.A. Volkenandt M. Flaig M. Yeung R.S. Wienecke R. Loss of tuberin, the tuberous-sclerosis-complex-2 gene product is associated with angiogenesis.J Cutan Pathol. 2001; 28: 470-475Crossref PubMed Scopus (36) Google Scholar, 22El-Hashemite N. Walker V. Zhang H. Kwiatkowski D.J. Loss of Tsc1 or Tsc2 induces vascular endothelial growth factor production through mammalian target of rapamycin.Cancer Res. 2003; 63: 5173-5177PubMed Google Scholar Brain and kidney lesions in TSC exhibit abnormally enhanced expression of the vascular endothelium protein marker CD31.23Arbiser J.L. Brat D. Hunter S. D'Armiento J. Henske E.P. Arbiser Z.K. Bai X. Goldberg G. Cohen C. Weiss S.W. Tuberous sclerosis-associated lesions of the kidney, brain, and skin are angiogenic neoplasms.J Am Acad Dermatol. 2002; 46: 376-380Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar Altered VEGF isoform D levels were observed in serum from TSC patients with lymphangioleiomyomatosis.24McCormack F.X. Lymphangioleiomyomatosis: a clinical update.Chest. 2008; 133: 507-516Crossref PubMed Scopus (250) Google Scholar Of note, enhanced VEGF expression in TSC may occur via both mTOR-dependent and mTOR-independent mechanisms.25Brugarolas J.B. Vazquez F. Reddy A. Sellers W.R. Kaelin Jr, W.G. TSC2 regulates VEGF through mTOR-dependent and -independent pathways.Cancer Cell. 2003; 4: 147-158Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar Altered expression of EGF, HGF, VEGF, and their receptors EGFR, c-Met, and Flt-1 (VEGFR1) has not been investigated in the brain in TSC, yet considerable evidence suggests that these factors play a pivotal role in the mTOR cascade's influence on cellular phenotype and could therefore provide insight into the mechanism of TSC cortical pathogenesis. Thus, we assayed both mRNA and protein expression in the Tsc1 conditional knockout (Tsc1GFAPCKO) mouse and in human TSC brain tissue specimens as a strategy to identify growth factors that could be targeted for therapeutic development in TSC. Tsc1GFAPCKO mice were generated as previously described26Uhlmann E.J. Wong M. Baldwin R.L. Bajenaru M.L. Onda H. Kwiatkowski D.J. Yamada K. Gutmann D.H. Astrocyte-specific TSC1 conditional knockout mice exhibit abnormal neuronal organization and seizures.Ann Neurol. 2002; 52: 285-296Crossref PubMed Scopus (285) Google Scholar at the Washington University School of Medicine in accordance with the guidelines established by the Animal Studies Committee of Washington University. Samples at postnatal days 1 and 10 were analyzed for several reasons. First, at these time points, the active phases of embryonic neuronal migration are completed; thus, there is homeostatic expression of growth factors and receptors. Second, by postnatal days 1 and 10 there is active expression of the Cre transgene in the brain; thus, adequate knockout of Tsc1 could be documented. Third, we wanted to be certain that none of the altered growth factor expression was a consequence of early seizures and altered behavioral phenotypes that occur in these animals by postnatal day 20. Poly(A) mRNA was extracted from the cerebral cortex of Tsc1GFAPCKO or wild-type (Wt) mice (n = 5 each) as described previously.15Kyin R. Hua Y. Baybis M. Scheithauer B. Kolson D. Uhlmann E. Gutmann D. Crino P.B. Differential cellular expression of neurotrophins in cortical tubers of the tuberous sclerosis complex.Am J Pathol. 2001; 159: 1541-1554Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar The entire cortex from both hemispheres was removed from the subcortical structures using a microscalpel at the level of the midhippocampus. Poly(A) mRNA served as a template for in vitro cDNA synthesis with avian myeloblastosis virus reverse transcriptase, and then double-stranded template cDNA was synthesized with T4 DNA polymerase I (Boehringer-Mannheim) from extracted cDNA. mRNA was amplified from the double-stranded cDNA with T7 RNA polymerase (Epicenter Technologies) incorporating 32P CTP as a radiolabel. Amplified mRNA served as a template for a second round of cDNA synthesis with avian myeloblastosis virus reverse transcriptase, deoxynucleotide triphosphates, and N6 random hexamers (Boehringer-Mannheim). cDNA generated from amplified mRNA was made double stranded and served as template for a second mRNA amplification, again incorporating 32P CTP radiolabel. The radiolabeled, amplified mRNA was used as a probe for cDNA arrays. cDNA arrays containing full-length mouse EGF, EGFR, HGF, c-Met, VEGF, and Flt-1 cDNAs were probed with 32P CTP–radiolabeled mRNA amplified from the cortex (one probe per array). All hybridization reactions were performed twice for each probe. Glyceraldehyde-3-phosphate dehydrogenase cDNA was included to serve as a positive hybridization control, and pBlueScript plasmid cDNAs were used to define background levels of hybridization on each array. Prehybridization (8 hours) and hybridization (24 hours) conditions were in 6× SSPE buffer, 5× Denhardt's solution, 50% formamide, 0.1% SDS, and salmon sperm DNA, 200 μg/ml, at 42°C. Blots were washed in 2× standard saline citrate. mRNA probe hybridization to array cDNAs was determined by phosphorimaging and densitometry. Tuber and SEGA samples (n = 15 patients; seven females; 10 tubers; six SEGAs; mean age, 9.9 years; in one patient a tuber and SEGA were removed en bloc; Table 1) were obtained from patients with clinically diagnosed TSC. There were no significant differences in age distribution in the TSC patient specimens used in the study. Tubers were removed as part of surgery for the treatment of intractable epilepsy, and SEGAs were removed to alleviate symptomatic hydrocephalus. Surgical tissue specimens were obtained from the Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, and Minnesota Epilepsy Group, St. Paul, MN. Surgical localization of the tuber resection site reflected the seizure focus as determined by scalp or intracranial electroencephalographic monitoring. SEGAs were identified preoperatively by magnetic resonance imaging as non-neurologic, progressively enlarging lesions that exhibited enhancement with gadolinium. Three SENs were obtained post mortem from three TSC patients (Table 1) who died of nonneurologic causes. In these three specimens, morphologically normal cortex adjacent to histologically defined tubers was also obtained. Clinical mutation testing (indicated as “genotype” in Table 1) results among the tuber, SEN, and SEGA patients revealed a TSC1 mutation in five patients and a TSC2 mutation in 10 patients; mutation data were unavailable from three patients (indicated as “NMI” in Table 1).Table 1Patient DemographicsSampleAge, yearsLocationGenotypeTuber4FrontalTSC1Tuber9FrontalTSC2Tuber3TemporalTSC2Tuber9FrontalTSC2Tuber3TemporalTSC1Tuber2TemporalTSC1Tuber4FrontalNMITuber6TemporalTSC2Tuber7FrontalNMITuber⁎Specimen contained both SEGA and cortical tuber.10FrontalTSC2SEGA⁎Specimen contained both SEGA and cortical tuber.10Lat ventTSC2SEGA14Lat ventTSC1SEGA21Lat ventTSC2SEGA22Lat ventTSC1SEGA19Lat ventTSC2SEGA16Lat ventNMISEN27Lat ventTSC2SEN37Lat ventTSC2SEN41Lat ventTSC2Epilepsy control6TemporalNMIEpilepsy control14TemporalNMIEpilepsy control12TemporalNMIEpilepsy control9TemporalNMIEpilepsy control16TemporalNMIControl4FrontalNMIControl9FrontalNMIControl11TemporalNMIControl11FrontalNMITable depicts age at time of surgery, lobar location of resection, and TSC genotype.Lat vent, lateral ventricle; NMI, no mutation identified. Specimen contained both SEGA and cortical tuber. Open table in a new tab Table depicts age at time of surgery, lobar location of resection, and TSC genotype. Lat vent, lateral ventricle; NMI, no mutation identified. Postmortem control brain tissue specimens (n = 4; two females; Table 1) were procured from the Brain and Tissue Bank for Developmental Disorders, University of Maryland (Baltimore, MD), from individuals who died of nonneurologic causes. Seizures were not terminal events in these patients, and none had a personal or family history of epilepsy or TSC. The cytoarchitecture of these specimens was intact. Additional surgical epilepsy control tissue consisted of temporal neocortical specimens (n = 5; three females; Table 1) obtained from individuals undergoing temporal lobectomy for intractable complex partial seizures (University of Pennsylvania Medical Center). These patients had no history or clinical findings compatible with a diagnosis of TSC, and the histologic features of the tissue samples were intact (these specimens were classified as epilepsy controls). All human tissue was obtained in accordance with protocols approved by the University of Pennsylvania Institutional Review Board and Committee on Human Research. All mouse and human tissue samples were immersion fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 8 μm. All fixed tissue blocks were hydrated through graded ethanols. Slides were pretreated with antigen unmasking solution (Vector Labs, Burlingame, CA) before immunostaining reactions. Sections were probed with one of the following antibodies: EGF (1:1000 dilution, rabbit polyclonal; Santa Cruz, Burlingame, CA), EGFR (1:100, rat polyclonal; Santa Cruz), EGFRvIII variant (courtesy of D. O'Rourke, M.D., Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA), phospho-EGFR (phospho Y1068, 1:250, rabbit monoclonal; Abcam, Cambridge, MA), HGF (1:500, mouse monoclonal; Abcam), c-Met (1:10, rabbit polyclonal; Abcam), VEGF (1:50, mouse monoclonal; Abcam), Flt-1 (1:100, mouse monoclonal; Santa Cruz), HIF-1α (1:1500, mouse monoclonal; Abcam), phospho-S6 ribosomal protein (p-S6, Ser235/236; 1:100, rabbit polyclonal; Cell Signaling, New England Biolabs, Beverly, MA), or S6 ribosomal protein (detects endogenous S6 expression, including both phosphorylated and nonphosphorylated isoforms, 1:100, rabbit monoclonal; Cell Signaling) overnight at 4°C and with secondary antibodies at room temperature for 1 hour. The slides were visualized using avidin-biotin conjugation (Vectastain ABC Elite; Vector Labs) with 3,3′-diaminobenzidine. After immunolabeling, sections were dehydrated through graded ethanols and xylene and coverslip mounted (Permount). Three representative contiguous digital photographs were obtained (20× magnification) from each mouse brain tissue section using image acquisition and analysis software (Spot RT CCD camera; Diagnostic Instruments Inc.; and Phase 3 Imaging System integrated with Image Pro Plus; Media Cybernetics, Silver Spring, MD). The three images spanned a 1-mm2 region of interest (ROI) within the lateral neocortex that was operationally defined and standardized across all cortex specimens as dorsolateral cerebral cortex at the level of the rostral hippocampus midway between the superior sagittal sulcus and the rhinal sulcus (Bregma coordinate −1.70 mm). The area of the cortex for each ROI was determined with a glass micrometer under light microscopy. In the human specimens, we were particularly interested in the number of GCs that expressed each protein growth factor marker. Thus, GCs were defined using maximal cell diameter based on cresyl violet and H&E staining for quantitative cell counting analysis. Representative digital photographs were obtained (magnification, ×20) under light microscopy from each tissue section (n = 3 sections per case) using image acquisition and analysis software as above. Each image spanned a 1-cm2 ROI. Before final assignment as a GC by the software, each ROI was visually inspected and cellular elements erroneously included in the computerized analysis were deleted. Mean maximal diameter (cell diameter at its largest aspect) was calculated using Image Pro Plus software as expressed in pixel units that were converted to microns by direct calibration with a micrometer. The relative optical density ratio (ODR) of labeled cells was calculated using Image Pro Plus software using a previously defined approach.15Kyin R. Hua Y. Baybis M. Scheithauer B. Kolson D. Uhlmann E. Gutmann D. Crino P.B. Differential cellular expression of neurotrophins in cortical tubers of the tuberous sclerosis complex.Am J Pathol. 2001; 159: 1541-1554Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar The ODR is calculated by determining the level of pixel staining density in labeled cells versus the pixel density of the noncellular background (the cell densities are digitally subtracted from the image). An ODR greater than 3 was used as a threshold to define immunopositivity for a given antibody. In the mouse, the total numbers of p-S6–immunolabeled cells were determined for each case and then expressed as a mean ± SEM for Tsc1GFAPCKO and W+ specimens. For growth factors, we used a semiquantitative scale (0 indicating no staining to ++++ indicating intense labeling across most cells in each ROI) to represent labeling density in each ROI. The size of p-S6–immunolabeled cells was defined using maximum cell diameter and determining the mean ± SEM). In the human specimens, the total numbers of morphologically identified GCs were determined in each ROI for each case, and the mean ± SEM numbers of GCs in ROIs were determined across all 10 tubers and 6 SEGAs. Statistically significant differences in GCs expressing individual protein markers were determined by Student's t-test (P < 0.05). Lysates of Wt and Tsc1GFAPCKO cortex were analyzed for p-S6 protein levels. A DuPont Kinetic Microplate Reader was used to approximate 15 μg of total protein for each of the samples, which were individually loaded into separate wells of a 4% to 15% Tris-HCl polyacrylamide gel (Bio-Rad Laboratories) and electrophoresed at 60 V. Proteins were then transferred overnight at 4°C onto a polyvinylidene difluoride (Millipore) membrane. Membranes were incubated in a 5% nonfat dry milk blocking solution for 1 hour at room temperature (RT) and then probed with rabbit anti–p-S6 ribosomal protein (Ser235/236; 1:1000, overnight at 4°C; Cell Signaling) antibodies. Rabbit anti–glyceraldehyde-3-phosphate dehydrogenase (1:1000, 1 hour at RT; Cell Signaling) and rabbit anti–β-actin (1:1000, 1 hour at RT; Cell Signaling) served as protein loading controls. Membranes were then incubated for 1 hour at RT with horseradish peroxidase–conjugated donkey anti-rabbit IgG (1:3000; GE Health Care). Membranes were washed and developed using either electrochemiluminescence or electrochemiluminescence plus Western blotting detection reagents (Amersham, GE Health Care) as needed for horseradish peroxidase visualization. The expression of each mRNA was determined by analysis of the radiolabeled mRNA-cDNA hybridization intensity on each array using ImageQuant5.0 software. Nonspecific hybridization to pBlueScript plasmid cDNA was subtracted from the hybridization intensity of each mRNA-cDNA to define specific hybridization intensity. The relative hybridization intensity for each mRNA was determined by averaging the phosphorimaging density of all of the mRNA-cDNA hybrids on each individual array and then expressing each mRNA-cDNA hybrid as a percentage of the average hybridization intensity of the entire array. Differences in relative mRNA abundance were determined using a one-way analysis of variance, and a Bonferroni post hoc correction was applied to each univariate analysis of variance. If a significant difference was found with a Bonferroni-adjusted analysis of variance, individual post hoc comparisons were made using the Fisher's test (P < 0.05 was considered significant). The expression level of Tsc1 mRNA was determined at postnatal days 1 and 10 in the cerebral cortex in Tsc1GFAPCKO mice. At postnatal day 1, Tsc1 mRNA levels were reduced by 86% ± 4%, and at postnatal day 10, levels were reduced by 92% ± 7% compared with Wt mice (n = 10 sections each in Tsc1GFAPCKO and Wt control samples at each time point, P < 0.05; Figure 1C). Expression of Tsc2 mRNA at postnatal days 1 and 10 in cortex from the Tsc1GFAPCKO mice did not differ from that observed in Wt mice. There was a low level of baseline p-S6 protein expression in cortical neurons and astrocytes of control Wt mice at postnatal days 1 and 10. p-S6–labeled astrocytes were observed throughout all cortical layers. In neurons, p-S6 expression was observed primarily within the somatic and dendritic cytoplasm of pyramidal cells in layers III and V. At both postnatal days 1 and 10, there was a clear increase in the number of p-S6–labeled cortical cells in the Tsc1GFAPCKO mouse brain compared with control Wt brains (Figure 1A). Quantitative cell counts of p-S6–labeled cells were performed at postnatal days 1 and 10, which antedates the onset of clinical seizures in these mice. There was a significant increase in the number of p-S6–labeled cells at postnatal day 1 (115 WT control and 336 Tsc1GFAPCKO mice, P < 0.05) and postnatal day 10 (665 control and 1319 Tsc1GFAPCKO mice, P < 0.05) (Figure 1B). Western assay revealed markedly enhanced S6 protein phosphorylation in Tsc1GFAPCKO mice compared with Wt control (Figure 1C).27Zeng L.H. Xu L. Gutmann D.H. Wong M. Rapamycin prevents epilepsy in a mouse model of tuberous sclerosis complex.Ann Neurol. 2008; 63: 444-453Crossref PubMed Scopus (483) Google Scholar Immunolabeling of human TSC cortex also revealed an increase in phosphorylation of S6, relative to control cortex, with no corresponding increase in endogenous S6 (both phosphorylated and nonphosphorylated isoforms) expression overall (Supplemental Figure 1, see http://ajp.amjpathol.org). Increased S6 phosphorylation in tubers is consistent with previous findings in TSC and confirms mTOR hyperactivity in these lesions.28Baybis M. Yu J. Lee A. Golden J.A. Weiner H. McKhann 2nd, G. Aronica E. Crino P.B. mTOR cascade activation distinguishes tubers from focal cortical dysplasia.Ann Neurol. 2004; 56: 478-487Crossref PubMed Scopus (221) Google Scholar The expression of EGF, EGFR, HGF, c-Met, and VEGF mRNAs in cerebral cortex was increased at postnatal days 1 and 10 in Tsc1GFAPCKO mice compared with Wt mice (Figure 2). EGFR mRNA levels were increased 9.3-fold and 7.1-fold at postnatal days 1 and 10, respectively, whereas EGF was increased 2.1-fold and 2.8-fold, HGF was increased 3.3-fold and fourfold, c-Met was increased threefold at both time points, and VEGF was increased 5.8-fold and 5.9-fold at postnatal days 1 and 10, respectively. However, Flt-1 mRNA expression in Tsc1GFAPCKO mice did not differ from that in Wt mice of either time point (data not shown). Of note, GFAP mRNA ex
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