| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Advances in Brief |
Gastroenterology Division [O. G. O., A. K. R.], Cancer Center [A. K. R.], Department of Genetics [A. K. R.], University of Pennsylvania, Philadelphia, Pennsylvania 19104
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
|
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Basal cells are subjected to a tightly regulated program of differentiation as they migrate toward the surface accompanied by a series of morphological, biochemical, and genetic changes. Insights into the biochemical and molecular genetic mechanisms that orchestrate the switch from proliferation to early differentiation in this cell type can be achieved through an understanding of the K3 genes that modulate this process and their transcriptional regulation. In this context, K5 and K14 heterodimerize in proliferating basal cells. The relatively ubiquitous K1 and K10 are linked to early differentiation genes and heterodimerize in suprabasal cells, whereas loricrin, profillagrin, and transglutaminase are among the late differentiation genes in superficial squamous cells (1 , 2) . Of significance, the suprabasal K4 and K13 are relatively tissue restricted, with highest expression in esophageal and corneal squamous epithelial cells (3) .
We previously generated a model in which K4 is disrupted through homologous recombination in murine embryonic stem cells, resulting in impaired differentiation and basal cell hyperplasia, specifically in esophageal and corneal epithelia of homozygous null mice (4) . This formed the basis for studying transcriptional regulatory mechanisms of the human K4 promoter in esophageal and corneal squamous epithelial cells. Previously, we have demonstrated that, in esophageal squamous epithelial cells, the human K4 promoter is transcriptionally regulated by esophageal-specific transcription factors (5) . To further elucidate how the regulation of K4 is linked to differentiation, we undertook analysis of its 5' untranslated regulatory region and promoter in corneal epithelial cells. Given that one of the roles of Sp1 is to modulate cellular differentiation and influence cell cycle progression in cooperation with the retinoblastoma protein (pRB), we focused on putative Sp1 DNA binding motifs in the K4 promoter and the role of Sp1 in regulating the K4 promoter. This was further investigated by comparing Sp1-mediated transactivation of the K4 promoter in parental normal corneal epithelial cells and stably transfected corneal epithelial cells with the cell cycle regulator, cyclin D1, the latter achieved through retroviral transduction. We describe herein that Sp1 transactivation of the K4 promoter in corneal epithelial cells is suppressed by cyclin D1, and that this can be rescued by concurrent ectopic expression of pRB. In particular, we demonstrate that cyclin D1 interacts with Sp1 in vivo, suggesting perhaps a model in which Sp1 may influence differentiation through interactions with either pRB or cyclin D1.
|
Materials and Methods |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
The amphotrophic packaging cell line Phoenix A was grown in supplemented DMEM (Sigma) and transiently transfected with the respective retroviral vector to generate amphotrophic retroviruses. Fresh retroviral supernatant was used for infection of exponentially growing SIRC cells. Several clones were harvested for further processing after puromycin selection. The retroviral expression vectors pBPSTR-D1 and pBabe-lacZ were obtained from S. Reeves (Massachusetts General Hospital, Charlestown, MA). The retroviral vector pBPSTR-D1 is described previously (6) and contains both elements of the tetracycline-regulated system. The retroviral expression vector pBPSTR is based on the vector pBabe, which accounts for comparable infectivity. The puromycin resistance gene under the control of the promoter within the 5' long terminal repeat is present in both retroviral vectors.
Transient Transfection.
Transient transfection of different K4 promoter deletion-luciferase
reporter constructs (K4-940, K4-540, K4-163, K4-140, and K4-76; Ref.
5
) and cotransfections of expression plasmids
pRC/RSV-empty, pRC/CMV-Sp1, pSG5-RB (wild-type pRB), pJ3 RB-592 (mutant
pRB), and pJ3 RB-209 (mutant pRB) were carried out using the calcium
phosphate precipitation technique (5'
3', Inc.), as described
previously (5
, 7
, 8)
. Incubations were performed in
triplicate, and results were calculated as the mean ± SE values for luciferase activity. Values were then expressed as fold
increase or decrease compared with the control for each set of
experiments. Activities were expressed as the mean of at least three
independent transfection experiments. Transfection efficiency was
controlled by cotransfection with pGreen Lantern-1 (Life Technologies,
Inc.) and found not to vary within a given transfection experiment,
indicating that transfection efficiency was uniform.
Flow Cytometry.
Exponentially growing cells were collected, and the cell pellet fixed
overnight in 70% ethanol and then resuspended in a 1-ml solution
containing 3.8 mM sodium citrate and 10 µg/ml propidium
iodide. After 10 mg/ml RNase treatment at 37°C for 20 min, the
samples were analyzed by a fluorescence-activated cell sorter (FACScan;
Becton Dickinson).
Coimmunoprecipitation and Western Blot Analysis.
Lysates from exponentially growing cells were harvested in a buffer
[50 mM HEPES (pH 7.4), 0.1% NP40, and 250 mM
NaCl] with 1 mM protease and 10 mM phosphatase
inhibitors. Total protein (150 µg) was incubated with 1 µg of
primary antibody at 4°C overnight, followed by incubation with
Protein A/Protein G Plus Agarose (Santa Cruz Biotechnology) for 2 h. Proteins were separated on 6-12% SDS-polyacrylamide gels and
transferred to Immobilon membranes (Millipore). Incubation with primary
antibodies was performed as indicated (1:3000). The secondary antibody
was either peroxidase-conjugated antimouse or antirabbit immunoglobulin
(Amersham Corp.; 1:2500). Detection was by chemiluminescence (ECL;
Amersham Corp.).
EMSAs.
Nuclear extracts from SIRC and C3D1 cell lines were prepared, and EMSAs
were performed as described previously (5
, 7
, 8)
using
probes Sp1-A (5'-AGCTT AACGG GTGCG GGAAG GATGG CTTGC-3'),
Sp1-C (5'-AGCTT AGGCT AAGGC TGGAC-3'), and Sp1-consensus
(5'-AGCTT ATTCG ATCGG GGCGG GGCGA GCC-3'). For
competition experiments, the nuclear extract was preincubated with
50-fold excess of unlabeled double-stranded Sp1 consensus
oligonucleotide prior to the addition of the
32P-labeled oligonucleotide probe. AP1
consensus oligonucleotide was used as a control. Immune supershift
assays were performed using a polyclonal anti-Sp1 (PEP2) or anti-AP1
antibody (D; Santa Cruz Biotechnology). The antibody was preincubated
with the nuclear extract at room temperature for 30 min prior to the
addition of the 32P-labeled oligonucleotide
DNA probe.
IP-EMSA.
IPs were done as described. Protein-agarose complexes were then washed
three times in gelshift buffer (7
, 8)
treated with 32 µl
of 0.8% deoxycholate to dissociate protein complexes on ice and
neutralized with 1% NP40. Ten µl of the supernatant were then used
for EMSAs as described above.
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
). Ectopic cyclin D1 was induced in asynchronously growing
cells in the absence of tetracycline. Of note, the levels of cyclin D1
in the presence of tetracycline were essentially the same observed in
the parental cell line (data not shown). Flow cytometry analysis
revealed pronounced cell cycle redistribution in C3D1 cells with a 20%
increase in cells cycling in S-phase compared with parental SIRC cells
(Fig. 1B
). Furthermore, C3D1 cells demonstrated an altered
morphology and grew in clusters with smaller size and rounded
appearance compared with the pleomorphic parental SIRC cells (data not
shown).
|
10-fold, which is
reduced by 50% in the K4-540, K4-163, and K4-140 deletion constructs
and almost abolished in the K4-76 deletion construct. These two
reductions in Sp1-mediated transactivation are consistent with two
distinct functional Sp1 cis-regulatory elements within the
K4 promoter, one within the K4-940 construct and the other one within
the K4-140 construct, respectively (Fig. 2A
). One putative Sp1 binding site resides at -649 bp and the
other one at -125 bp.
|
).
Because pRB is the major substrate of cyclin D1 and is known to act as
a transcriptional activator through Sp1 (11
, 12)
, the role
of pRB role in influencing the transcriptional regulation of K4 was
assessed. Interestingly, cotransfection with wild-type pRB, but not
mutated pRB (pRB-592 and pRB-209), resulted in a 10-fold
transactivation of the K4 promoter in C3D1 cells (Fig. 2B
).
Thus, pRB rescues the reduction in Sp1-mediated transactivation of the
K4 promoter in cyclin D1-overexpressing cells. It has been described
previously that pRB regulates the transcription of target genes through
cis-acting elements referred to as retinoblastoma control
elements, which can bind members of the Sp1 family (11
, 13) . In contrast, pRB cotransfection showed only a 5-fold
transactivation of the K4 promoter in parental SIRC cells (Fig. 2B
). Cotransfection of pRB and Sp1 did not cause any
significant additional increase in K4 promoter activity in both cell
lines (Fig. 2B
).
Sp1 Binds to cis Regulatory Elements of the Human K4
Promoter.
To examine whether the putative Sp1 cis-regulatory elements
indeed bind Sp1, we performed EMSAs (5
, 7
, 8)
with either
SIRC or C3D1 nuclear extracts. EMSAs were performed with two
double-stranded oligonucleotide probes containing the putative Sp1
elements within the K4 promoter, residing at positions -649 bp (Sp1-C)
and -125 bp (Sp1-A), respectively. As control, a Sp1 consensus
oligonucleotide probe was used. One DNA-protein complex was observed
with Sp1-A, Sp1-C, and Sp1-consensus oligonucleotide probes, suggesting
that these DNA-protein complexes were formed by Sp1 and an
oligonucleotide harboring an Sp1 DNA binding motif (Fig. 2C
). Adding 50-fold excess of unlabeled Sp1 consensus
oligonucleotide (Fig. 2D
) specifically inhibited Sp1-DNA
complex formation. Human Sp1-specific antibody further decreased the
Sp1-DNA complex (Fig. 2D
). AP1 oligonucleotide and antibody
served as controls.
Comparison of Sp1-DNA complexes in both cell lines indicated a decrease
with C3D1 nuclear extracts compared with SIRC nuclear extracts, thereby
suggesting diminished DNA binding by Sp1 in cyclin D1-overexpressing
cells (Fig. 2D
). These results indicate that cyclin D1
overexpression influences the transcriptional regulation of the K4
promoter in corneal epithelial cells by interfering with the function
of Sp1 as a transcriptional activator.
Differential Expression of pRB and Sp1 in Cyclin D1-overexpressing
Corneal Epithelial Cells.
To clarify differences in Sp1-DNA complex formation in C3D1 cells
versus SIRC cells, we determined Sp1 and pRB protein levels
in these cell lines. Western blot analysis of pRB levels, using a pRB
antibody that equally recognizes hypo- as well as hyperphosphorylated
pRB, revealed a shift toward the hyperphosphorylated form of pRB in
C3D1 cells (Fig. 3A
), consistent with cyclin D1 overexpression. Western blot
analysis of Sp1 revealed no appreciable differences between C3D1 and
SIRC cells (Fig. 3B
).
|
). We found that Sp1 physically interacted
with either pRB or cyclin D1. Whereas no differences in levels between
the two cell lines could be determined with the recently described
Sp1-pRB interaction (Ref. 12
; Fig. 4A
), a more
prominent Sp1-cyclin D1 interaction was detected in C3D1 cells compared
with SIRC cells (Fig. 4B
). However, in C3D1 cells, Sp1
interacts predominantly with hyperphosphorylated pRB, whereas in SIRC
cells an interaction with both forms of pRB could be observed (Fig. 4A
). As a negative control, J82 cells, where pRB is
truncated, and DT40 cells, where cyclin D1 is deleted, were
immunoprecipitated with Sp1 antibody and immunoblotted with either
anti-pRB antibody (Fig. 4A
) or anti-cyclin D1 antibody (Fig. 4B
), respectively.
|
C, Lanes 5 and 6)
or cyclin D1 immunoprecipitates (Fig. 4
C, Lanes 9 and
10). However, a more prominent DNA-protein complex could be
observed in immunoprecipitates from C3D1 cells. Addition of
Sp1-specific antibody, but not AP1 antibody, attenuated this
immunoprecipitated Sp1 complex (Fig. 4C
). Sp1
immunoprecipitates (Fig. 4
C, Lanes 1 and 2) and
AP1 immunoprecipitates (Fig. 4
C, Lanes 15 and 16)
served as positive or negative controls, respectively. In aggregate,
our findings are consistent with an interaction between Sp1 and
cyclin D1 that leads to differential regulation of a
differentiation-linked K4 promoter. |
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Indeed, a natural question emanating from these findings was whether cyclin D1 and Sp1 interacted with each other apart from the recently described Sp1-pRB interaction (12) . Our data are consistent with the notion that cyclin D1 and Sp1 do interact. It is tempting to speculate that the Sp1-pRB complex may mediate transactivation of genes, the protein products of which in turn favor growth suppression and/or commit the cell to early differentiation. The role of Sp1 in differentiation or growth suppression is further evidenced by its interaction with Smad proteins to regulate the p21/Cip1/WAF1 promoter (15) . Nonetheless, the functional effects of pRb-Sp1 may be opposed by the Sp1-cyclin D1 complex, a consequence of which is to retard or partially disrupt the commitment to differentiation. Certainly, such a phenomenon would be evident in an exaggerated form in some tumor types, for example squamous cell cancers, which frequently overexpress cyclin D1. It should be emphasized that we were able to detect the Sp1-cyclin D1 interaction in normal corneal epithelial cells, indicating that this is not the result of cyclin D1 overexpression in the C3D1 cells. However, the Sp1-cyclin D1 interaction is more dramatic in C3D1 cells with apparent functional consequences of diminished Sp1-mediated transactivation of the K4 promoter. Mapping of the domains involved in the Sp1-cyclin D1 interaction will be of interest.
Our data do not preclude the possibility that the cyclin D1-Sp1 complex may be part of a larger complex that regulates transcription, and indeed, one such candidate may be TFIID because members of this family have been demonstrated recently to interact individually with cyclin D1, Sp1, or pRB (16, 17, 18) . It is also conceivable that cyclin D1 phosphorylates Sp1 because phosphorylation of Sp1 has been shown to be important in cell cycle regulation of its own transcriptional activity (19) . Additionally, the cyclin D1-Sp1 complex may also contain pRb, a possibility not excluded by our experiments. Whether cyclin D1-Sp1 complex formation is dependent upon cdk4/cdk6, as with the phosphorylation of pRB, or independent of cdk activity as is the case with the cyclin D1-estrogen receptor interaction (20) , requires further investigation. Whereas the ability of Sp1 to influence the equilibrium between proliferation and differentiation is mediated in part through its interactions with cell cycle regulatory proteins, cyclin D1 in turn may promote the tendency to proliferation through interaction with proteins such as estrogen receptor in breast epithelial cells and Sp1 in corneal epithelial cells and possibly, in different cell types.
|
Acknowledgments |
|---|
|
FOOTNOTES |
|---|
1 This work was supported by NIH Grants
R01-DK53377 (to A. R.), P01-DE12467 (to A. R. and O. O.), American
Cancer Society Jr. Faculty Research Award JFRA-649 (to A. R.), The
Leonard and Madlyn Abramson Family Cancer Research Institute (to
A. R.), and Deutsche Krebshilfe Grant D/96/17197 (to O. O.). 
2 To whom requests for reprints should be
addressed, at Gastroenterology Division, 600A CRB, University of
Pennsylvania, 415 Curie Boulevard, Philadelphia, PA 19104. Phone:
(215) 898-0154; Fax: (215) 573-5412; E-mail: anil2{at}mail.med.upenn.edu
3 The abbreviations used are: K, keratin; EMSA,
electrophoretic mobility shift assay; IP, immunoprecipitation; cdk,
cyclin-dependent kinase.
Received 12/ 1/99. Accepted 4/10/00.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  N. Y. Tan and L. M. Khachigian Sp1 Phosphorylation and Its Regulation of Gene Transcription Mol. Cell. Biol., May 15, 2009; 29(10): 2483 - 2488. [Full Text] [PDF]
|
|
|
|
 |
|
 J. Pujal, M. Huch, A. Jose, I. Abasolo, A. Rodolosse, A. Duch, L. Sanchez-Palazon, F. J. D. Smith, W. H. I. McLean, C. Fillat, et al. Keratin 7 promoter selectively targets transgene expression to normal and neoplastic pancreatic ductal cells in vitro and in vivo FASEB J, May 1, 2009; 23(5): 1366 - 1375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Adhikary, J. F. Crish, R. Gopalakrishnan, F. Bone, and R. L. Eckert Involucrin Expression in the Corneal Epithelium: An Essential Role for Sp1 Transcription Factors Invest. Ophthalmol. Vis. Sci., September 1, 2005; 46(9): 3109 - 3120. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Adhikary, J. F. Crish, F. Bone, R. Gopalakrishnan, J. Lass, and R. L. Eckert An Involucrin Promoter AP1 Transcription Factor Binding Site Is Required for Expression of Involucrin in the Corneal Epithelium In Vivo Invest. Ophthalmol. Vis. Sci., April 1, 2005; 46(4): 1219 - 1227. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Adhikary, J. Crish, J. Lass, and R. L. Eckert Regulation of Involucrin Expression in Normal Human Corneal Epithelial Cells: A Role for Activator Protein One Invest. Ophthalmol. Vis. Sci., April 1, 2004; 45(4): 1080 - 1087. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-E. Gingras, K. Larouche, N. Larouche, S. Leclerc, C. Salesse, and S. L. Guerin Regulation of the Integrin Subunit {alpha}5 Gene Promoter by the Transcription Factors Sp1/Sp3 Is Influenced by the Cell Density in Rabbit Corneal Epithelial Cells Invest. Ophthalmol. Vis. Sci., September 1, 2003; 44(9): 3742 - 3755. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Boudreau, E. H. H. M. Rings, H. M. van Wering, R. K. Kim, G. P. Swain, S. D. Krasinski, J. Moffett, R. J. Grand, E. R. Suh, and P. G. Traber Hepatocyte Nuclear Factor-1alpha , GATA-4, and Caudal Related Homeodomain Protein Cdx2 Interact Functionally to Modulate Intestinal Gene Transcription. IMPLICATION FOR THE DEVELOPMENTAL REGULATION OF THE SUCRASE-ISOMALTASE GENE J. Biol. Chem., August 23, 2002; 277(35): 31909 - 31917. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Gheorghiu, C. Deschenes, M. Blais, F. Boudreau, N. Rivard, and C. Asselin Role of Specific CCAAT/Enhancer-binding Protein Isoforms in Intestinal Epithelial Cells J. Biol. Chem., November 16, 2001; 276(47): 44331 - 44337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Aslam, L. Palumbo, L. H. Augenlicht, and A. Velcich The Sp Family of Transcription Factors in the Regulation of the Human and Mouse MUC2 Gene Promoters Cancer Res., January 1, 2001; 61(2): 570 - 576. [Abstract] [Full Text]
|
|
|
|
|
|
F. H. Brembeck and A. K. Rustgi The Tissue-dependent Keratin 19 Gene Transcription Is Regulated by GKLF/KLF4 and Sp1 J. Biol. Chem., September 1, 2000; 275(36): 28230 - 28239. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
