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Departments of Cell Biology and Physiology The University of
Pittsburgh School of Medicine Pittsburgh, Pennsylvania 15261
Magee-Womens Research Institute Department of Obstetrics,
Gynecology and Reproductive Sciences University of Pittsburgh
School of Medicine Pittsburgh, Pennsylvania 15213
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We demonstrated previously that the expression of the cAMP-dependent transcription factor CREB (cAMP response element-binding protein) is extinguished upon the luteinization of granulosa cells in the monkey ovary and proposed that the loss of CREB signaling might subtract the expression of a subset of genes involved in the control of cellular proliferation and survival (7). We reasoned that the consequences of the loss of CREB in luteal cells could be determined by eliminating the CREB-signaling pathway in progenitor granulosa cells. To accomplish this, we used a recombinant adenovirus vector that directs the expression of a mutant of CREB (CREB M1) in which the activating phosphorylation site at serine 133 is mutated to alanine (8). Herein we show that while interfering with CREB signaling in granulosa cells does not inhibit their differentiation into steroid-producing cells, expression of CREB-M1 adversely affects their viability. These findings suggest that CREB may function as a molecular switch that governs cell survival in the ovary and support the rapidly emerging notion that CREB may function as a survival factor in a number of different cell types.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, Ad ß-gal-directed ß-galactosidase
activity in primary cultures of immature rat granulosa cells was
hormone responsive. In the absence of hormonal stimulation (panel A),
ß-galactosidase staining was weak. In contrast, incubation of
granulosa cells with either 10 ng/ml human (h)FSH (panel B) or 10
µM forskolin (FSK) (panel D) resulted in a more
intense staining of individual cells. The expression of
ß-galactosidase was ligand-dependent as immature granulosa cells that
lack LH receptors did not respond to the addition of 10 ng/ml hLH
(panel C).
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(top
panel) illustrates a Western immunoblot using an antibody directed
against the amino terminus of CREB on whole-cell lysates of granulosa
cells that were infected with increasing amounts of the Ad CREB M1
adenovirus. Exposure of granulosa cells to increasing viral titers
resulted in progressive increases in immunoreactive CREB expression as
compared with noninfected cells. When cell lysates from noninfected
granulosa cells were subjected to immunoprecipitation with an anti-CREB
antibody and the immunoprecipitates exposed to protein kinase A (PKA)
and [32P]-ATP in vitro, intense
phosphorylation of a 43-kDA protein was observed (Fig. 2, bottom
panel). In contrast, immunoprecipitates of granulosa cells from Ad
CREB M1-infected cells were weakly phosphorylated by PKA in
vitro as would be expected by the elimination of the PKA-dependent
phosphorylation site on ser 133.
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shows that Ad CREB M1
inhibited FSH-stimulated CMV-ß-galactosidase activity in a
dose-dependent fashion, with the highest Ad CREB M1 concentration
(1:100 dilution) completely inhibiting ß-galactosidase expression.
This concentration of Ad-CREB also inhibited ß-galactosidase
expression in response to 10 µM FSK and 0.5
mM 8-Br-cAMP (data not shown).
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). The
observation that ß-galactosidase activity was somewhat lower in Ad
EGFP-infected cells in comparison to noninfected cells may be due to
competition of the CMV promoters for endogenous transcriptional
regulators, as both basal and FSK-stimulated ß-galactosidase
activities were reduced proportionately. The relative increases in
ß-galactosidase activity in response to FSK were comparable in cells
not exposed to virus and those exposed to Ad EGFP (18-fold
vs. 13-fold, respectively).
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illustrates that estrogen production after 48 h exposure
to FSH or FSK was comparable by cells not exposed to adenovirus and
cells exposed to Ad EGFP. The responses to FSK were statistically
significant (P < 0.05, n = 4 independent
observations) when compared with untreated cells. Although cells
exposed to Ad CREB M1 produced lower amounts of estrogen when compared
with noninfected cells or Ad EGFP-infected cells, they retained their
responsiveness to FSH and FSK (P < 0.05). For all
groups, there was a net increase in estrogen concentration between
samples collected at 24 and 48 h after exposure to FSH or FSK
(data not shown). Identical results were observed with progesterone
production (Fig. 4C).
Effect of Ad CREB M1 on Granulosa Cell Survival
Figure 5 illustrates the morphology
of the granulosa cells from the study shown in Fig. 4. The top
panel presents fluorescent microscopic analysis in the granulosa
cells infected with Ad EGFP. Like that of the CMV-ß-gal, the
expression of the CMV promoter-directed EGFP was FSH and FSK
responsive. The center panel illustrates the morphology of
Ad EGFP-infected granulosa cells. Well defined monolayers were present
in cells not exposed to stimuli as well as cells exposed to FSH or FSK.
The lower panel illustrates the morphology of granulosa
cells exposed to Ad CREB M1. In the absence of hormone stimulation or
in the presence of FSH, granulosa cells were present in well defined
monolayers. In marked contrast, Ad CREB M1-infected granulosa cells
that were exposed to 10 µM FSK displayed pronounced
morphological alterations that included profound cell clumping, which
was associated with the detachment of the majority of the cells from
the culture dish.
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. There were no significant differences
in the viability of cells (P > 0.05) exposed to Ad
EGFP compared with cells not exposed to virus either in the presence or
absence of FSK. Although not apparent by visual examination, granulosa
cells infected with Ad CREB M1 and not exposed to FSK exhibited a
significant (P < 0.01) decline in viability at 48 and
72 h when compared with cells not infected with virus or cells
infected with Ad EGFP. At both 48 h and 72 h, incubation of
Ad CREB M1-infected cells with 10 µM FSK resulted in a
further decrease in viability when compared with Ad CREB M1-infected
cells not exposed to FSK (P < 0.01). The reduced
production of estrogen and progesterone in Ad CREB M1-infected
granulosa cells in response to FSK (Fig. 4, B and C) is thus likely due
to a diminished number of viable cells in the culture.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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s
are associated with elevated levels of phosphorylated CREB (13). CREB was first identified as a nuclear protein that is phosphorylated by PKA and binds to the cAMP response element (CRE) on the somatostatin gene promoter and stimulates transcription (14). Since its identification as a PKA-dependent transcriptional activator, it has become apparent that CREB is a nuclear target of a number of intracellular signaling pathways including mitogen-activated protein kinase (15), p90RSK (16), and calmodulin kinases (17, 18) as well as the antiapoptotic Akt/PKB pathway (19). Given the wide diversity of inputs that may converge on CREB, it is not unreasonable to suggest that CREB may be involved in basic processes of cellular homeostasis including proliferation and cell death. Our current finding that expression of CREB M1 results in diminished viability of granulosa cells is consistent with other recent studies indicating that CREB may function as a survival factor. Thus, in addition to causing cell cycle arrest in T cells, the expression of a dominant-negative mutant of CREB in transgenic animals also resulted in apoptosis of these cells in response to agents that would normally stimulate activation (10). Likewise, expression of a dominant-negative mutant of CREB in human melanoma cells led to reduced tumorigenesis as well as an enhanced sensitivity of these cells to apoptosis (20). Whether the diminished viability of granulosa cells as assessed by the MTT assay seen in the current study is the result of apoptotic cell death is not known and will be the subject of future research.
During the course of our studies, we observed that the expression of ß-galactosidase under the control of a CMV promoter was highly responsive to FSH, FSK, and cAMP stimulation. In retrospect, the responsiveness of the CMV promoter to cAMP is not surprising as it has been shown by others that the CMV promoter contains three cAMP response elements (CREs) and is stimulated by FSK (21). Using the hormonal dependence of CMV-ß-galactosidase as a reporter for documenting the effectiveness of CREB M1, we demonstrated that FSH- and FSK-stimulated ß-galactosidase expression was completely blocked by overexpression of the mutant CREB. Assuming that there are no differences between the ability of CREB M1 to inhibit the transcription of the extrachromosomal CMV-ß-galactosidase gene and endogenous genes, we would conclude that CREB-mediated expression of endogenous genes was dramatically compromised in these cells. Despite the apparent inhibition of CREB-dependent transcription, estradiol and progesterone production by CREB M1-expressing granulosa cells remained highly responsive to FSH and FSK. Although we have not measured mRNA levels for steroidogenic enzymes in the present study, it is well recognized that the acquisition of the ability of granulosa cells from immature rats to produce estrogen and progesterone is due to cAMP-mediated increases in mRNAs for aromatase (P450arom), cholesterol side chain cleavage (P450scc), and 3ß-hydroxysteroid dehydrogenase, 5–4 isomerase (3ß-HSD) (22). We would therefore infer from our studies that those genes involved in progesterone and estrogen production are not CREB dependent. This is consistent with the findings of others that the promoter regions of P450scc and 3ß-HSD do not appear to contain CREs (23, 24) and that P450arom, which does contain a CRE-like sequence, also appears to be responsive to steroidogenic factor-1 (25). The separation of cAMP-regulated signaling pathways that control survival from those that control differentiated phenotype (steroidogenesis) in the ovary would serve to ensure that progesterone production, which is essential for the maintenance of pregnancy, continues even though trophic actions of cAMP are lost after ovulation.
A number of potential mechanisms exist by which the elimination of
CREB-mediated signaling in granulosa cells may adversely affect cell
proliferation and survival. CREB may act as a transcription factor that
directly regulates genes involved in these processes. One candidate
gene is proliferating cell nuclear antigen (PCNA), an auxiliary factor
for DNA polymerase 
, which is required for both replicative and
repair DNA synthesis, as both the murine and the human PCNA gene
promoters contain obligatory CREs (26, 27). Indeed, in the primate
ovary, the expression of both PCNA and CREB cease upon luteinization
(7). A second possible candidate gene is cyclin D2, which has been
shown to be FSH and FSK responsive in rat granulosa cells (28, 29).
Interestingly, ovarian function in the cyclin D2 knockout mouse is
similar to that seen in our current study as granulosa cell
proliferation, but not granulosa cell differentiation
(steroidogenesis), is disrupted (28). It is not yet known, however,
whether expression of the cyclin D2 gene is CREB dependent.
Alternately, CREB could also act by controlling the expression of other
transcription factors that regulate genes involved in proliferation and
survival such as c-fos and C/EBPß, both of which are
CREB-dependent (30, 31). In this regard it is noteworthy that the
C/EBPß knockout mouse is infertile due to defects in the
luteinization process (32).
In our current study, pronounced morphological disruptions and reduced
granulosa cell viability were most apparent when Ad CREB M1-infected
cells were stimulated by FSK. This observation is analogous to those
obtained with other cells in which the expression of inactive CREB
mutants resulted in diminished cell viability. Thus, Jean et
al. (20) did not observe marked cytopathic effects of a CREB
mutant on melanoma cells unless the cells were treated with
thapsigargin, which elevates intracellular free Ca++
concentrations. Likewise, Barton et al. (10) did not observe
pronounced cell death in T cells that express a nonphosphorylatable
CREB mutant unless the cells were presented with activating stimuli. As
FSK has been shown to elevate intracellular free Ca++ in
granulosa cells (33), it is possible that the elimination of
CREB-mediated signaling allows other pathways that are antagonistic to
cell proliferation and survival to exert dominance, possibly by
increasing their ability to interact with limited amounts of nuclear
coactivators (34). In this regard, previous studies by Aharoni
et al. (35) demonstrated that treatment of granulosa cells
from rat preovulatory follicles with very high concentrations of FSK
(50 µM) promoted apoptosis but, like that seen in our
study, did not inhibit steroid production. As we did not observe any
cytotoxic effects of a lower concentration (10 µM) of FSK
in noninfected cells or cells infected with either Ad ßgal or Ad EGFP
(Figs. 1D, 5 and 6), it appears that the loss of CREB signaling
rendered granulosa cells more sensitive to adverse effects of FSK, as
would be expected if competition exists between CREB and other
antagonistic signaling pathways.
A caveat to the interpretation of our current results is the assumption that the cytopathic effect of CREB M1 on granulosa cells is due specifically to the competition of this mutant protein with endogenous CREB for binding to CREs within the regulatory regions of CREB-responsive genes. However, it remains a possibility that overexpression of CREB M1 in granulosa cells may squelch the activity of other signaling pathways. For example, in addition to forming homodimers, CREB is also able to form heterodimers with other members of the bZip family of transcription factors, such as ATF-1 (36). Thus, an alternate explanation for our findings could be that the effects of CREB M1 could indirectly be mediated through sequestration of ATF 1 or other transcription factors. Although we cannot rule this out, our previous studies with the primate ovary failed to detect, by Southwestern analysis, any CRE-binding proteins other than CREB in luteal cells (7). In addition, CREB has been shown to interact in a phosphorylation-independent manner with components of the TFIID complex (37). However, such an interaction with the general transcription factor apparatus would be expected to globally suppress transcription, which would be inconsistent with our findings that CREB M1 did not block granulosa cell differentiation. Finally, it is important to note that cytopathic effects of CREB M1 expression were not observed in either ovarian epithelial cancer cells (as presented in Results) or primary cultures of rat Sertoli cells (9). The latter is significant in view of the fact that Sertoli cells, which are the testicular homologs of granulosa cells, do not require FSH-mediated cAMP signaling for their survival in vivo (38).
In summary, our current results suggest that CREB may function as a molecular switch that governs cell proliferation and survival in the ovary, results that are in keeping with other recent observations that CREB may function as a general regulator of cell proliferation and survival (8, 9, 10, 11, 12, 13, 19, 20). Extrapolation of these observations to the in vivo state suggests that the loss of CREB expression that occurs in the primate corpus luteum could be directly causal to the cessation of proliferation of luteal cells and, ultimately, the cell death that occurs during luteal regression.
The recent report that phosphorylated CREB is absent from nuclei of rat luteal cells indicates that an impairment in CREB signaling also occurs in rodents (39). Although the downstream targets of CREB that may participate in cell proliferation and survival are not known, the ability to rapidly and effectively interfere with CREB-mediated signaling in primary cell cultures with replication-defective adenovirus vectors provides a novel approach to address this important problem.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The methods for generating and propagating recombinant adenoviruses are described in detail elsewhere (40). In brief, 10 µg of the plasmid pACsk.2CMV CREB M1 were cotransfected with 10 µg of the plasmid pJM17, a plasmid containing a circularized adenovirus type 5 (variant dl309) genome (40) into the human embryonic kidney cell line 293 (41) using a calcium phosphate transfection system according to the manufacturer’s instructions (Life Technologies, Inc., Gaithersburg, MD). After transfection the cells were maintained in DMEM containing 4.5 g/liter glucose (Life Technologies, Inc.) and 10% FBS at 37 C in 5% CO2 for 13 days at which time the cells exhibited viral cytopathic effect. Cells and tissue culture supernatants were collected, frozen on dry ice and thawed three times, and then centrifuged (1000 x g, 4 C, 10 min) to remove cellular debris. Aliquots of virus stocks were diluted 50- and 100-fold in lysis solution [0.1% SDS, 10 mM Tris-Cl (pH 7.4), 1 mM EDTA] and incubated for 10 min at 56 C in a shaking water bath. The absorbance of the samples was measured at 260 nm, and the value obtained was used to calculate virus content using the equation 1.0 absorbance units = 1.1 x 1012 virus particles/ml (42). Adenoviruses were propagated by infecting 293 cells with approximately 108 particles/ml in tissue culture medium without serum. Infected cells were incubated until they exhibited nearly complete cytopathic effect and processed as above. Virus stocks were prepared to a concentration of 6 x 1012 particles per ml as described above and diluted for use as indicated in Results.
Granulosa Cell Culture and Adenovirus Infection
All procedures were approved by the Magee-Womens Research
Institute Institutional Animal Use and Care Committee. Immature female
rats (20 or 25 days old) were purchased from Taconic Farms, Inc. (Germantown, NY) and were housed under standard husbandry
conditions. For studies shown in Fig. 2, animals received a
subcutaneously placed 1-cm SILASTIC capsule containing crystalline
diethylstilbestrol (Sigma Chemical Co., St. Louis,
MO) on day 20 of age and were killed on day 25 of age. The remainder of
the studies used granulosa cells from untreated 22- to 25-day-old rats.
Granulosa cells were collected from the ovaries by puncturing follicles
with a 25-gauge hypodermic needle, and cells were expressed into Medium
199 (M199; Life Technologies, Inc.) containing 10% FBS.
Granulosa cells were seeded into 6-well (
106 cells per
well) or 24-well (2 x 105 cells per well) tissue
culture plates and allowed to attach overnight. The next morning,
medium and unattached cells were removed and the granulosa cell
monolayers were exposed to adenoviruses in M199 without protein
supplements for 2 h at 37 C with occasional rocking. Medium was
replaced with fresh M199 containing 1 mg/ml BSA (Sigma Chemical Co.). Twenty-four hours after exposure to adenoviruses, medium
was removed and replaced with M199 plus BSA also containing 10 ng/ml
testosterone (Sigma Chemical Co.) alone or with addition
of human FSH (AFP 4161B; 3205 IU FSH/mg < 225 IU LH/mg) or FSK
(Sigma Chemical Co.).
Immunoblot Analysis of Lysates from AdCREB M1-Infected Primary
Rat Granulosa Cell Cultures
Granulosa cells were harvested by scraping into ice-cold PBS
followed by centrifugation (16,000 x g, 4 C, 10 min).
Pelleted cells were resuspended in Western lysis buffer (50
mM Tris-Cl, pH 7.4, 1 mM EDTA, 20 µg/ml
phenylmethylsulfonylfluoride, 0.5 µg/ml leupeptin, 0.7 µg/ml
Pepstatin A, 10 nM Microcystin LR), lysed by sonication,
and processed for anti-CREB immunoblotting as described previously (7)
using a rabbit anti-rat CREB antibody directed against the first 205
amino acids of the amino-terminal region of CREB (Upstate Biotechnology, Inc., Lake Placid, NY).
Immunoprecipitation and in Vitro PKA
Phosphorylation
Ad CREB M1-infected and control granulosa cells were harvested
in ice-cold RIPA buffer [150 mM NaCl, 10 mM
Tris-Cl (pH 7.5), 0.1% (wt/vol) SDS, 1% (vol/vol) NP-40]
supplemented with protease and phosphatase inhibitors (20 µg/ml
phenylmethylsulfonylfluoride, 0.5 µg/ml leupeptin, 10 nM
Microcystin LR, 200 µM sodium vanadate).
Immunoprecipitation of CREB was performed using the antibody directed
against the amino-terminal region of CREB as described above, and
in vitro phosphorylation of immunoprecipitates by PKA were
performed as described previously (43).
ß-Galactosidase Assays
Identification of ß-galactosidase-expressing cells in
granulosa cell cultures was assessed by histochemistry using X-gal as
substrate (42). Granulosa cell monolayers were fixed in 2%
paraformaldehyde for 60 min, washed three times in PBS, and incubated
in Xgal solution containing 2 mM
K3Fe(CN)6, 2 mM
K4Fe(CN)6, 1 mM MgCl2,
and 1 mg/ml Xgal. Quantification of ß-galactosidase activity was
performed by fluorometric analysis (44). Granulosa cells were harvested
by scraping into ice-cold PBS, and centrifuged (16,000 x
g, 4 C, 10 min) to pellet the cells. The PBS was removed,
and the cell pellets were frozen on dry ice and then stored at -80 C
until use. Cell pellets were thawed on ice with the addition of 100
µl ß-galactosidase assay buffer (150 mM Tris-Cl, pH
7.5, 10 mM MgCl2, 25 mM NaCl, 10
mM 2-mercaptoethanol). Identical volumes of cell extract
were added to a reaction mixture containing 150 mM Tris-Cl,
pH 7.5, 10 mM MgCl2, 25 mM NaCl, 10
mM 2-mercaptoethanol, 150 nM
4-methylumbelliferyl ß-galactoside, and 1 mg/ml BSA and incubated at
30 C for 30 min, after which reactions were stopped by the addition of
0.5 ml of 0.25 M glycine (pH 10.65). Three hundred
microliters of each assay were transferred to a 96-well dish, and
fluorescence was measured using an excitation wavelength of 340 nm and
emission wavelength of 460 nm.
Steroid Production
Estradiol and progesterone concentrations of culture medium were
determined by RIAs as described previously (45).
Cell Viability Assay
Viability of granulosa cells was assessed using the CellTiter
assay (Promega Corp., Madison WI) in which a tetrazolium
salt is bioreduced into a formazan by metabolically active cells.
Assays were performed according to the manufacturer’s direction and
were terminated 4 h after the addition of substrate, at which time
the production of the formazan, measured by recording the absorbance at
490 nm, was linear as a function of time.
Statistics
Results were assessed for statistical significance by ANOVA
followed by comparison of group means with Duncans multiple range
analyses (46).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by a Johnson & Johnson Focused Giving Award and NIH Grant HD-16842 (A.J.Z.).
Received for publication February 2, 1999. Revision received May 3, 1999. Accepted for publication May 13, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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N. Kaivo-Oja, J. Bondestam, M. Kamarainen, J. Koskimies, U. Vitt, M. Cranfield, K. Vuojolainen, J. P. Kallio, V. M. Olkkonen, M. Hayashi, et al. Growth Differentiation Factor-9 Induces Smad2 Activation and Inhibin B Production in Cultured Human Granulosa-Luteal Cells J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 755 - 762. [Abstract] [Full Text] [PDF]
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 Y. Funakoshi, T. Ichiki, K. Takeda, T. Tokuno, N. Iino, and A. Takeshita Critical Role of cAMP-response Element-binding Protein for Angiotensin II-induced Hypertrophy of Vascular Smooth Muscle Cells J. Biol. Chem., May 17, 2002; 277(21): 18710 - 18717. [Abstract] [Full Text] [PDF]
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S. C. Bleckmann, J. A. Blendy, D. Rudolph, A. P. Monaghan, W. Schmid, and G. Schutz Activating Transcription Factor 1 and CREB Are Important for Cell Survival during Early Mouse Development Mol. Cell. Biol., March 15, 2002; 22(6): 1919 - 1925. [Abstract] [Full Text] [PDF]
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P. R. Manna, M. T. Dyson, D. W. Eubank, B. J. Clark, E. Lalli, P. Sassone-Corsi, A. J. Zeleznik, and D. M. Stocco Regulation of Steroidogenesis and the Steroidogenic Acute Regulatory Protein by a Member of the cAMP Response-Element Binding Protein Family Mol. Endocrinol., January 1, 2002; 16(1): 184 - 199. [Abstract] [Full Text] [PDF]
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T. Tokunou, T. Ichiki, K. Takeda, Y. Funakoshi, N. Iino, H. Shimokawa, K. Egashira, and A. Takeshita Thrombin Induces Interleukin-6 Expression Through the cAMP Response Element in Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, November 1, 2001; 21(11): 1759 - 1763. [Abstract] [Full Text] [PDF]
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T. Tokunou, T. Ichiki, K. Takeda, Y. Funakoshi, N. Iino, and A. Takeshita cAMP Response Element-Binding Protein Mediates Thrombin-Induced Proliferation of Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, November 1, 2001; 21(11): 1764 - 1769. [Abstract] [Full Text] [PDF]
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Z. Bebia, J. P. Somers, G. Liu, L. Ihrig, A. Shenker, and A. J. Zeleznik Adenovirus-Directed Expression of Functional Luteinizing Hormone (LH) Receptors in Undifferentiated Rat Granulosa Cells: Evidence for Differential Signaling through Follicle-Stimulating Hormone and LH Receptors Endocrinology, June 1, 2001; 142(6): 2252 - 2259. [Abstract] [Full Text] [PDF]
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M. J. Scobey, S. Bertera, J. P. Somers, S. C. Watkins, A. J. Zeleznik, and W. H. Walker Delivery of a Cyclic Adenosine 3',5'-Monophosphate Response Element-Binding Protein (CREB) Mutant to Seminiferous Tubules Results in Impaired Spermatogenesis Endocrinology, February 1, 2001; 142(2): 948 - 954. [Abstract] [Full Text] [PDF]
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 A. Riccio, S. Ahn, C. M. Davenport, J. A. Blendy, and D. D. Ginty Mediation by a CREB Family Transcription Factor of NGF-Dependent Survival of Sympathetic Neurons Science, December 17, 1999; 286(5448): 2358 - 2361. [Abstract] [Full Text]
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A. Bonni, A. Brunet, A. E. West, S. R. Datta, M. A. Takasu, and M. E. Greenberg Cell Survival Promoted by the Ras-MAPK Signaling Pathway by Transcription-Dependent and -Independent Mechanisms Science, November 12, 1999; 286(5443): 1358 - 1362. [Abstract] [Full Text]
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N. Mitsuda, N. Ohkubo, M. Tamatani, Y.-D. Lee, M. Taniguchi, K. Namikawa, H. Kiyama, A. Yamaguchi, N. Sato, K. Sakata, et al. Activated cAMP-response Element-binding Protein Regulates Neuronal Expression of Presenilin-1 J. Biol. Chem., March 23, 2001; 276(13): 9688 - 9698. [Abstract] [Full Text] [PDF]
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