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Laboratory of Molecular Endocrinology, Massachusetts General Hospital, Howard Hughes Medical Institute and Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Joel F. Habener, M.D., Laboratory of Molecular Endocrinology, Massachusetts General Hospital, 55 Fruit Street, Wellman 306, Boston, Massachusetts 02114. E-mail: jhabener{at}partners.org.
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Introduction |
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Alzheimer’s disease is an age-related form of dementia due to a progressive loss of neuronal functioning, also characterized by the deposition of extracellular amyloid throughout the brain (amyloid plaques) and a significant decrease in brain mass. An overactivity of the cyclin-dependent protein kinase CDK5 in the brain has been implicated in neuronal degeneration and the formation of neurofibrillary tangles due to hyperphosphorylation of essential neuronal proteins such as MAP and Tau (2). CDK5 is regulated by the neuron-specific and cyclin-related protein p35 (also known as p35Ncdk5a). During the development of Alzheimer’s disease, p35 is processed to a smaller protein, p25, which is a constitutive activator of CDK5. The generation of p25 disrupts CDK5 localization and substrate preferences (2). In addition, the expression of p25 in neurons results in a collapse of microtubules, neurite retraction, and apoptosis (2).
Despite their endodermal origin, pancreatic endocrine cells share many molecular and cellular characteristics with neural ectoderm-derived neurons. Several specialized genes are expressed in both the nervous system and insulin-producing ß-cells of the pancreas. Genes such as enolase, tetanus toxin receptor, and the A2B5 antigen initially found in the nervous system were later found to be expressed in ß-cells (3). Another group of genes normally expressed in islet of Langerhans has been shown to be present in the nervous system. Some of these genes are only temporally expressed in the developing brain, such as insulin and the transcription factor IDX-1 (islet and duodenum homeobox-1) [PDX-1 (pancreatic and duodenal homeobox-1)] (4, 5, 6, 7), whereas other genes are permanently expressed, such as the genes for ATP-sensitive K+ channel (8, 9) and the LIM (Lin-11, Isl-1, and Mec-3) homeodomain protein islet-1 (10).
Because of the similarities between neurons and pancreatic ß-cells and between the neural degeneration of Alzheimer’s disease and the deterioration of ß-cell functioning in adipogenic diabetes, we reasoned that p35/CDK5 may also be expressed in ß-cells and contribute to the pathogenesis of diabetes mellitus. This idea was reinforced by the recent findings of statistical correlations between Alzheimer-type dementia and diabetes mellitus in longitudinal population-based cohort studies (11, 12), and the fact that the yeast ortholog of CDK5, Pho85, is involved in metabolic control (13, 14). We further hypothesized that continuous elevation of serum glucose may dysregulate CDK5 activity and thereby may provide an explanation of how persistent hyperglycemia may contribute to the progressive failure of ß-cell functioning in diabetes. To determine whether the p35/CDK5 has a possible functional role on ß-cell activity we examined p35/CDK5 in ß-cells. Our studies comprise mRNA detection by RT-PCR, cDNA cloning, and kinase activity assays. Furthermore, functional studies in cultured clonal INS-1 cells as well as experiments to assure specificity were performed. Our findings indicate that 1) pancreatic islets and cultured ß-cells express both p35 and CDK5; 2) elevations of the concentration of glucose in the culture medium result in induction of p35 expression that is translated into activation of the p35/CDK5 kinase activity; and 3) the expression of p35 and the activation of p35/CDK5 enhance transcriptional activation of the insulin gene. This effect on the insulin gene promoter is specific because it is prevented by roscovitine, an inhibitor of CDK5 activity, by a dominant negative form of CDK5, and by the use of small interfering RNA (siRNA), which acts by destabilizing the p35 mRNA and thereby reducing the level of p35 protein.
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Materials and Methods |
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Cell lines and tissue culture
Rat INS-1 cells (provided by C. Wollheim, Geneva, Switzerland; passages 99–110) were cultured in RPMI 1640 medium containing 10% fetal calf serum, 10 mmol/liter HEPES, 11.1 mmol/liter glucose, 1 mmol/liter sodium pyruvate, 50 µmol/liter 2ß-mercaptoethanol, 100 µU/ml penicillin G, and 100 mg/ml streptomycin. HeLa cells and NIH-3T3 fibroblasts were cultured in DMEM containing 10% fetal calf serum, 100 µU/ml penicillin G, and 100 mg/ml streptomycin. Cells were maintained at 37 C in a humidified incubator gassed with 5% CO2. Cell cultures were passaged by trypsinization and subcultured every 5 d.
Animals and human tissue sources
Male Wistar rats, 8–9 wk old and weighing 200–250 g (The Jackson Laboratory, Bar Harbor, ME), were obtained for preparation of pancreatic tissue sections for immunocytochemistry. Purified human islets were obtained from the islet distribution programs of Cell Transplant Center, Diabetes Research Institute, University of Miami School of Medicine (Miami, FL), and Juvenile Diabetes Foundation Center for Islet Transplantation, Harvard Medical School (Boston, MA). Thoroughly washed islets were handpicked before performing RNA isolation and protein extraction for Western blot and kinase assays. Animal experiments were conducted in accordance with NIH guidelines for the care and use of laboratory animals and with approval from the Massachusetts General Hospital institutional animal care and use committee.
Plasmid DNA constructs
A rat p35 expression plasmid was constructed by RT-PCR amplification of the rat p35 coding sequence from INS-1 cells total RNA, maintaining its own Kozak sequence and inserting it into the pCR3 vector by TA cloning. The integrity of the construct was verified by DNA sequencing and Western blot detection of the corresponding expressed protein after transfection. Once the sequence was confirmed the same amplified cDNA was inserted in-frame into the pCMV-tag2 vector (Stratagene, La Jolla, CA) containing a Flag tag in its amino-terminal domain. A similar approach was used to construct an expression plasmid for rat CDK5 and to create a dominant negative version of CDK5. The latter was generated by substituting the GAT codon at position 144 for AAT through the use of the QuikChange kit (Stratagene, La Jolla, CA) as previously described by Van den Heuvel and Harlow (15). The –410 rat insulin-1 promoter luciferase construct was provided by Dr. M. German (University of San Francisco, San Francisco, CA).
RT, amplification by PCR, and Southern blot analysis
Total cellular RNA (5 µg) dissolved in 10 µl H2O was combined with 0.5 µg oligo(deoxythymidine) (Life Technologies, Inc.) and heated to 65 C for 10 min, then cooled on ice. RT buffer, deoxy-NTPs (50 µM each), dithiothreitol (5 mM), H2O, and 50 U SuperScript II enzyme (Life Technologies, Inc.) were incubated at 42 C for 40 min in a 40-µl reaction. PCRs were carried out using 2 µl cDNA template, 20 pmol each of forward and reverse primers, 0.2 µM each of deoxy-NTPs, 1x buffer, and 2.5 U thermostable Taq polymerase (TaKaRa Biomedical, Inc., Berkeley, CA) in a 100-µl reaction volume. Amplifications were performed with 30 cycles of 30-sec denaturation at 95 C, 30-sec annealing at 55 C, and 1-min extension at 72 C. Sense orientation primer for the rat p35 were: RP35-1, 5'-CGCGCAGTGGCACCATGGGCACGGTGCTGTCCCTG-3'; and nested, RP35-12, 5'-TCCCCCAGCTATCGAAAGGCCACAC-3'. The sequence of the antisense primers was: RP35-2, 5'-GGAGCGGTCCACGCTGCGCAGCCAGAGCACGGGGT-3'; and nested: RP35-22, 5'-CGTTGGGGACAAGTGCTTCAGGCGG-3'. Confirmation of the identity of the amplified band was achieved by Southern blot analysis after transferring the amplified DNA onto a nitrocellulose filter and hybridization with a 32P-labeled internal oligonucleotide probe RP35-3 (5'-GGTTCTCATTGTTGAGGTGCGCGATGTTGCTCTGGTAGCTGCTGTTGGGCTGCGCCTTCTTGGAGTTC-3'). For the human p35 sequence, the sense primer was HP35–1 (5'-GCGCGCAGCAGCACCATGGGCACGGTGCTGTCC-3'), and the antisense primer was HP35-2 (5'-TGCTCACCGATCCAGGCCTAGGAGGAGCCGCTT-3').
Immunocytochemistry
Cryosections of 6 µm were prepared from rat pancreases and fixed with 4% paraformaldehyde in PBS. Sections were first blocked with 3% normal donkey serum for 45 min at room temperature. Subsequently, they were incubated with primary antibody at a 1:500 dilution overnight in a cold room (see Materials and Methods). The next day the sections were rinsed in PBS and incubated with the corresponding Cy3- and Cy2-labeled secondary donkey antisera for 1 h at room temperature. Slides were then washed with PBS and mounted using fluorescent mounting medium (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Fluorescent images were obtained using a epifluorescence microscope (Carl Zeiss, Inc., New York, NY) equipped with an TEC-470 CCD camera (Optronics International, Chelmsford, MA).
Immunoprecipitation and kinase assay
We followed the protocol of Nikolic and Tsai (16) for immunoprecipitation and detection of p35/CDK5 activity. Briefly, the cells or tissues were homogenized in lysis buffer A, containing 20 mM Tris-HCl (pH 7.2), 2 mM MgCl2, 0.5% Nonidet P-40, 150 mM NaCl, and 1 mM dithiothreitol. The following protease and phosphatase inhibitors were also added: phenylmethylsulfonylfluoride (1 mM), leupeptin (2 µg/ml), aprotinin (1 µg/ml), NaF (5 mM), and NaVO4 (1 mM). For p35 immunoprecipitation, cellular extracts containing 1.5 mg protein were incubated with 3 µl of the corresponding p35 (C-19) antibody at 4 C overnight and then with 100 µl protein A-Sepharose (10 mg/ml) for an additional hour at 4 C. The samples were washed three times with lysis buffer and used for determination of kinase activity after two additional washes in kinase buffer. The in vitro kinase reaction was performed at 30 C for 30 min in kinase buffer [50 mM HEPES (pH 7.0), 10 mM MgCl2, 5 mM MnCl2, and 1 mM dithiothreitol] containing 1 µCi [
-32P]ATP and 5 µg histone H1 in a 50-µl reaction volume. A equal volume of Laemmli loading buffer was added to stop the reaction, and samples were loaded in a 10% SDS-PAGE after boiling for 5 min. Gels were dried and exposed for autoradiography.
Western blot assays
Protein extracts were run in a 10% SDS-PAGE gel (200 µg protein/lane) and transferred onto a nitrocellulose membrane at 4 C overnight. Blocking was performed for 1 h in 10% nonfat milk and 0.1% Tween 20 at room temperature. The membranes were then incubated for an additional 1 h in 1% nonfat milk and 0.1% Tween 20 with the primary antibody at a 1:200 dilution for p35 and CDK5 antibody and a 1:1000 dilution for insulin antibody. An additional incubation with a horseradish peroxidase-conjugated secondary antibody was also performed at room temperature for 1 h. After three consecutive washes of 15 min each, the reactive proteins were developed with the the luminescent enhanced chemiluminescence system from Amersham Pharmacia Biotech (Arlington Heights, IL).
Transfection experiments and luciferase assays
Adherent INS-1 cells grown to 80–90% confluence in 28-well plates were transfected using Lipofectamine-2000 (Life Technologies, Inc.). Cells were rinsed twice in serum-free culture medium before the addition of the transfection cocktail containing 250 ng reporter plasmid DNA and 500 ng p35 expression plasmid (pCR3-p35). Cells were incubated in this mixture for 4 h before adding the normal growth medium and incubating them for an additional 48 h before harvesting for luciferase content assays. Roscovitine treatment was applied 24 h before harvesting the cells at the indicated concentrations. Firefly luciferase activity was determined using a luciferase assay kit (Promega Corp., Madison, WI). All experiments were carried out in triplicate on at least three independent occasions. Data (emitted light units) are expressed as the mean ± SE.
Inhibition of p35 expression with siRNA (double-stranded RNA) specific for p35
To specifically inhibit the expression of p35, we used small (21-bp) double-stranded RNAs designed with a sequence complementary to the p35 mRNA. These RNAs, also known as siRNA, were synthesized, HPLC-purified, annealed (Dharmacon Research, Inc., Lafayette, CO), and then cotransfected with the insulin promoter luciferase reporter (–410 RIP luciferase) and the expression vector for p35. The sequence of the sense RNA strand was 5'-GAGGAUCGUGGCGGUGUCATT-3', and that of the antisense sequence was 5'-UGACACCGCCACGAUCCUCTT-3'. An additional siRNA with a nonrelated sequence was used as a control. To test for the effectiveness of this siRNA, it was cotransfected in the same cells with a Flag-tagged p35 construct, and the corresponding protein was detected by Western blot analysis (see Fig. 5C
). The final concentration of dsRNA during transfection was 200 nM, as recommended by the manufacturer. The addition of fresh medium after 4 h diluted the RNA interference to 100 nM. This concentration was maintained for 48 h until cells were harvested.
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Results |
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). Both p35 and CDK5 immunostaining colocalized with insulin staining in double-stained sections (Fig. 1A). The pattern of staining was heterogeneous among different islets. The larger pancreatic islets showed greater p35 and CDK5 staining intensities compared with the smaller islets. Most of the small islets showed weak or negative staining. To confirm the presence of p35 and CDK5 in pancreatic islets of Langerhans, we used isolated human islets to investigate the presence of the specific proteins and mRNAs by Western blot analysis and RT-PCR, respectively. A specific amplified DNA product was obtained by RT-PCR. This product showed the predicted size when analyzed by electrophoresis in a 1% agarose gel, and its identity was confirmed by Southern blot analysis (Fig. 1B) and by direct sequencing of the DNA after cloning in a TA pCRII system (data not shown). To detect a protein product for p35 and CDK5, isolated human islets of Langerhans were homogenized in buffer A (see Materials and Methods) and equivalent aliquots (200 µg protein) of mouse brain (positive control) and liver (negative control) were analyzed by Western blot. A p35-immunoreactive band was detected in the islet extract that comigrated with a control p35 protein in extract of mouse brain (Fig. 1C). This protein was not present in the liver extract that served as a negative control (Fig. 1C). After reblotting the membrane with a CDK5 antibody, widespread expression of CDK5 protein was also detected in human islets as well as in brain and liver extracts (Fig. 1C).
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). No kinase activity was detected in the absence of antibody or in the presence of an unrelated control antibody (Fig. 1D).
Expression of CDK5 and its activator p35 in ß-cells correlates with the presence of specific p35/CDK5 kinase activity
Additional experiments were designed to investigate the presence of p35, CDK5, and p35/CDK5 kinase activity in ß-cell lines. Biochemical characterization of CDK5 kinase activity was also investigated. Among the different cell lines tested, only INS-1 cells, an insulin-producing ß-cell line, showed expression of p35 (Fig. 2A). These cells also contain protein kinase activity that can be immunoprecipitated with a p35-specific antibody (Fig. 2B). Both p35 expression and p35/CDK5 activity were absent in other cell lines, such as HeLa and NIH-3T3 (Fig. 2, A and B), although these cell lines expressed CDK5 protein (Fig. 2A). We then investigated whether the kinase activity immunoprecipitated by the p35 antibody was due to its association with CDK5. We performed experiments with roscovitine, a relatively specific inhibitor of CDK5 activity (17). The p35/CDK5 activity in INS-1 cells was inhibited by roscovitine, but not by other protein kinase inhibitors, such as H89 (protein kinase A) and SB202190 (p38MAPK; Fig. 2C). Also, the inhibition by roscovitine was dose dependent (Fig. 2D), with a 50% inhibitory concentration of 128 nM, within the range of that previously reported for CDK5 (18).
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). An immunoprecipitate obtained with an antibody reactive toward an unrelated protein (control) did not show kinase activity. The activity detected with the CDK5 antibody was weaker than that obtained with an equivalent amount of p35 antibody in both INS-1 cells and brain extracts. As both p35 and CDK5 antibodies were equally active in extracts from INS-1 and mouse brain, we considered several possibilities to explain the differences between the two antibodies. First, the CDK5 antibody may have a lower affinity for its protein target. Second, CDK5 antibody binding to the p35/CDK5 complex may partially interfere with the functional activity of the complex. Finally, as CDK5 protein is in excess with respect to p35, active p35/CDK5 complexes not only may be immunoprecipitated by the CDK5 antibody, but also may uncouple CDK5 without kinase activity. To provide further evidence that the immunoprecipitated kinase activity depends on the presence of CDK5 we performed Western blots with the CDK5 antibody on the immunoprecipitated material. Results from these experiments clearly indicated p35 and CDK5 associate in INS1 cells, resulting in the formation of a functionally active kinase complex (Fig. 3B).
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). We then evaluated the capacity of this expression vector to reconstitute an active functional p35/CDK5 kinase complex in INS-1 cells. As INS-1 cells already have endogenous p35/CDK5 activity, we used the expression vector coding for a p35 protein tagged with a Flag sequence and immunoprecipitated cellular extracts 48 h after transfection. Specific protein kinase activity was detected after immunoprecipitation with a specific Flag antibody, but not by using an unrelated control antibody (Fig. 3D).
The concentration of glucose in the culture medium regulates p35 expression and p35/CDK5 kinase activity in INS-1 cells
We next investigated whether the expression of the CDK5 activator p35 in ß-cells may itself be regulated by glucose. Our results indicate that the level of p35 mRNA in ß-cells is stimulated by the extracellular concentration of glucose. RT-PCR experiments show a progressive increase in the p35 mRNA content of INS-1 cells when they are exposed for 24 h to increasing concentrations of glucose in the culture medium (Fig. 4A). The increase in p35 mRNA levels is specific, because no changes were observed in the levels of ß-actin mRNA, which served as a control (Fig. 4A). The increase in p35 mRNA is translated into a corresponding increase in the levels of p35 protein as assessed by Western blot assays (Fig. 4B). No changes in the levels of CDK5 protein were observed in these experiments (Fig. 4B), indicating that the CDK5 regulatory subunit p35, and not the CDK5 protein itself, is regulated by glucose. To determine whether the increase in p35 resulted in a functional elevation of p35/CDK5 kinase activity, we performed immunoprecipitation and kinase assays. The results of these experiments show a corresponding elevation of p35/CDK5 activity when the extracellular concentration of glucose was increased from 5 to 20 mM at different time points ranging from 2–24 h (Fig. 4C). The time-dependent induction in p35/CDK5 kinase activity started between 1 and 2 h after glucose stimulation (Fig. 4D).
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) without affecting the expression of other proteins not targeted by the siRNA. The effect of p35-siRNA in the suppression of endogenous p35 was less effective. siRNA needs to enter the cells via transfection, and the efficiency of transfection in these experiments was approximately 20%. The cotransfection of an expression vector for p35 with a luciferase reporter under the control of the insulin gene promoter results in a 3-fold activation of transcription (Fig. 5B). Activation of the insulin promoter by p35 was significantly inhibited by cotransfection of a specific p35 siRNA as well as by the administration of roscovitine (10 µM), a CDK5-specific inhibitor (Fig. 5B). Similar results were obtained in several preliminary experiments when the efficiency of transfection was corrected by cotransfecting an expression vector for Renilla luciferase (data not shown). In these experiments no effect on luciferase activity was detected when an unrelated siRNA control was used. In addition, a dominant negative mutant of CDK5 (CDK5-DN) containing a point mutation that affects its active site (15) inhibited p35-stimulated insulin promoter activity (Fig. 5C). These observations indicate that the activation of the insulin promoter by p35 is specific and is a result of the formation of a functional p35/CDK5 complex with protein kinase activity. |
Discussion |
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Our findings demonstrate the expression of p35, the activator of CDK5, in insulin-producing ß-cells. Expression of p35 in the pancreas was not detected earlier in Northern blot testing for tissue-specific expression (19), probably due to the fact that only a small fraction of the total pancreas RNA is derived from islets. Also, the expression of p35 at a normal physiological concentration of glucose is very low in ß-cells, its maximal expression is only achieved after glucose stimulation. Our data also confirm the expression of CDK5 in ß-cells and demonstrate the existence of a functional p35/CDK5 protein kinase pathway in insulin-producing ß-cells.
Perhaps the most important finding in these studies is the fact that the expression of p35 and the protein kinase activity of the p35/CDK5 complex in ß-cells are tightly regulated by changes in the extracellular concentration of glucose. It is known that the expression of genes essential for the function of ß-cells, such as insulin, PDX-1 (pancreatic and duodenal homeobox-1), amylin, and glucose transporter 2, are closely regulated by extracellular concentrations of glucose. This circumstance suggests that the p35/CDK5 protein kinase pathway may play an important role in ß-cell function. In fact, our initial functional studies indicate that p35/CDK5 is involved in the expression of the insulin gene. Our data show that overexpression of p35 results in an increased activity of the insulin promoter in INS-1 cells, and that inhibition of the p35/CDK5 activity using several strategies, such as specific siRNA for p35; the use of roscovitine, a relatively specific CDK5 inhibitor (17); and cotransfection of a dominant negative mutant form of CDK5 (15), decreases the activation of the insulin promoter induced by p35.
In neurons, p35/CDK5 regulates not only cytoskeletal dynamics and cell migration, but also the cAMP-dependent protein kinase A and the calcium-mediated signaling pathway (14). It also regulates dopamine signaling through phosphorylation of DARPP-32, which converts it into an inhibitor of protein kinase A (25). Therefore, we anticipate that in ß-cells the presence of p35/CDK5 could be involved in the regulation of additional functional pathways. Our findings also indicate that in insulin-producing ß-cells, CDK5 activity is predominantly regulated by variations in the expression of its activator p35, which, in turn, is regulated by extracellular glucose concentrations. During brain development, the expression of p35 seems to be constitutive, and regulation of CDK5 activity is dependent on its subcellular localization, phosphorylation by other protein kinases such as Abl, and association with other regulatory proteins (Dab1 and Cables) (14). However, up-regulation of p35 at the level of transcription can also occur, as shown after stimulation by laminin in cultured neurons (26), nerve growth factor in PC12 cells (27), neuroregulin in cultured myotubules (23), and chronic cocaine administration to rats (28).
Our studies raise the possibility that overstimulation of the p35/CDK5 pathway by elevated glucose levels and mislocalization due to cleavage of p35 into a p25 form, lacking its myristoylation domain, may also be a pathogenic mechanism for the glucotoxicity of ß-cells. Overactivation and mislocalization of p35/CDK5 could translate the deleterious effect of a combination of different pathological signals, such as amylin deposition, high levels of plasma lipoproteins, and high glucose levels, as dysregulation of p35/CDK5 in the central nervous system has been associated with the pathological abnormalities found in Alzheimer’s disease (2, 29) and amyotrophic lateral sclerosis (30). It is possible that glucose-induced dysregulation of the p35/CDK5 pathway is a pathophysiological mechanism involved in the ß-cell dysfunction and the predisposition to apoptotic cell death associated with the progression of type 2 diabetes. Several population-based studies have shown that diabetes is associated not only with vascular dementia, but also with Alzheimer’s type dementia (11, 31, 32, 33). Furthermore, a recent longitudinal population-based cohort study of the Honolulu-Asia Aging Study has clearly demonstrated an association between type 2 diabetes and Alzheimer’s dementia and has shown that this association is significantly higher among carriers of the APOE 
4 allele (12). These and other interesting possibilities await the results of further experimental studies.
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Acknowledgments |
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Footnotes |
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Abbreviations: CDK, Cyclin-dependent protein kinase; siRNA, small interfering RNA.
Received November 11, 2003.
Accepted for publication February 9, 2004.
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