Expression of PDE11A in Normal and Malignant Human Tissues
Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, LLC, Spring House, Pennsylvania (MRDA), and Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, LLC, Raritan, New Jersey (YQ,DH-J,SB,PK,SL)
Correspondence to: Yuhong Qiu, Drug Discovery, Johnson & Johnson Pharmaceutical Research & Development, LLC, 1000 Route 202, Raritan, NJ 08869. E-mail: yqiu{at}prdus.jnj.com
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Summary |
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Key Words: PDE11A immunohistochemistry tissue distribution normal and cancer human tissues
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Introduction |
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PDE11A is the most recent PDE to be cloned. It is most similar to PDE5, based on sequence similarity, but unlike PDE5 it is a dual-substrate enzyme, hydrolyzing both cAMP and cGMP (Fawcett et al. 2000). Four splice variants have been identifiedPDE11A1, A2, A3, and A4that encode proteins of 490, 576, 684, and 943 amino acids, respectively (Fawcett et al. 2000
; Yuasa et al. 2000
; Hetman et al. 2000
). These predict molecular mass of 56, 66, 78, and 105 kDa for PDE11A1A4, respectively. The four isoforms contain the same carboxyl termini, whereas PDE11A2, A3, and A4 possess unique amino termini (Hetman et al. 2000
; Yuasa et al. 2000
). Analysis of the steady-state RNA levels have indicated that human PDE11A is most abundantly expressed in the prostate, followed by the testis, liver, skeletal muscle, pituitary gland, pancreas, and kidney (Fawcett et al. 2000
; Yuasa et al. 2000
). Although recent studies have provided Western blot analyses in some tissues (Fawcett et al. 2000
), the cellular localization of PDE11A is not known. We used immunohistochemistry (IHC) to detect PDE11A (all four splice variants) in a variety of normal and malignant human tissues showing its primary cellular localization in normal epithelial, smooth muscle, and endothelial cells, as well as in a variety of carcinoma cells.
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Materials and Methods |
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PDE Assay
The effects of several known PDE inhibitors on PDE11A4 were studied using PDE assays described previously (Qiu et al. 2003). Briefly, the assays were conducted in 5 mM MgCl2, 15 mM Tris HCl (pH 7.4), 0.5 mg/ml bovine serum albumin, 30 nM [3H] cGMP (Amersham Pharmacia Biotech; Piscataway, NJ). The total volume of the assay was 100 µl. The reaction mixture was incubated at 30C for 90 min and then stopped by boiling for 1 min, followed by cooling on ice. The resulting [3H] 5'-mononucleotides were further converted to uncharged [3H] nucleosides by adding 25 µl of 1 mg/ml snake venom (Ophiophagus hannah) and incubating at 30C for 10 min. This reaction was stopped by the addition of 1 ml AG1-X2 resin slurry (Bio-Rad; Hercules, CA). After centrifugation at 3000 x g, the [3H] in a 200-µl aliquot of the supernatant solution was quantified by liquid scintillation counting. The amount of PDE11A4 used was such that the hydrolysis of substrates in the peak fractions did not exceed 15% of the total, ensuring that the amount of product increased linearly with respect to time. Percent inhibition was used to generate IC50 values by fitting the data to a non-linear regression model in GraphPad Prism (Graphpad Software Inc.; San Diego, CA). Sildenafil was extracted from Viagra tablets (Pfizer; Groton, CT) and used as the citrate salt. All other PDE inhibitors were purchased from Sigma-Aldrich (St Louis, MO).
Western Blot Analysis
An affinity-purified rabbit polyclonal antibody (PD11A-112AP) was purchased from FabGennix, Inc. (Shreveport, LA). This antibody was generated against a sequence located at the C terminus (VATNRSKWEELHQKR) of human PDE11A that was common to all known splice variants. Human cytosolic proteins from various tissues processed as described by Boolell et al. (1996) were obtained from Analytical Biochemical Services, Inc. (Wilmington, DE). Hi5 cell lysates, human cytosolic proteins (100 µg/lane), and recombinant PDE11A4 were fractionated on 412% Bis-Tris SDS mini-gel (Invitrogen). Proteins were transferred to nitrocellulose membranes (0.45 µm; Invitrogen) using a mini-transblot system (Bio-Rad). The membrane was blocked with 5% blocking agent (Amersham Pharmacia Biotech) in TBS at 4C for 2.5 hr and probed PD11A-112AP at 1:500 dilutions in 1% blocking agent in TBS at 4C for overnight. The excess antibody was removed by washing the membrane in 1% blocking solution. Following the washes, alkaline phosphatase-conjugated sheep anti-rabbit IgG antibody diluted 1:10,000 in 1% blocking solution was added to the membrane and incubated for 2.5 hr at 4C. After washing off the excess secondary antibody, the blot was developed with the BCIP/NBT kit from Calbiochem (La Jolla, CA).
Immunohistochemistry
Normal (NormalGrid) and malignant (TumorGrid) human tissue slides were purchased from Biomeda (Foster City, CA). Table 2 lists all of the tissues present on this slide. Methods for IHC have been previously described (D'Andrea et al. 2003). Briefly, tissue sections on microscopic slides were dewaxed and re-hydrated and then processed for IHC. Slides were microwaved in Target buffer (DAKO; Carpinteria, CA), cooled, placed in phosphate-buffered saline (pH 7.4, PBS; Zymed Labs, South San Francisco, CA), and treated with 3.0% H2O2 for 10 min at room temperature. All incubations (30 min) and washes were performed at room temperature. Normal blocking serum (Vector Labs; Burlingame, CA) was placed on the slides for 10 min. After a rinse in PBS, sections were incubated with the rabbit anti-PDE11A antibodies (1:100; FabGennix, Inc.) We also applied the monoclonal anti-vimentin antibodies (DAKO) as a positive control for tissue antigenicity as well as the replacement of the primary antibody with the antibody dilution buffer as a negative control. Furthermore, we preabsorbed the anti-PDE11A with recombinant antigen (35x titer excess) overnight at 4C before placement on the slide as an additional control. Slides were then washed in PBS and treated with biotinylated secondary antibodies (Vector Labs). After washing in PBS, the avidinbiotinhorseradish peroxidase complex reagent (Vector Labs) was added. Slides were washed and were treated with 3,3'-diaminobenzidine (Biomeda) two times each for 5 min, rinsed in dH2O, and counterstained with hematoxylin.
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Results |
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Discussion |
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The antibody used here was raised against a peptide located at the C terminus. Thus, it should recognize all four splice variants. The identity of the recombinant protein was confirmed by enzyme activity in PDE assays and by its sensitivity to known PDE inhibitors. PDE11A4 has 943 amino acids with a predicted molecular mass of 105 kDa. The recombinant protein migrated at the expected location. The specificity of the antibody was confirmed using Western blot analysis on Hi5 cell lysates infected with expression vectors containing PDE11A4 cDNA or an unrelated cDNA. All human tissues on the Western blot displayed a band that migrated similarly as the recombinant PDE11A4, with different intensity in different tissues. This suggested that PDE11A4 was present in the tissues examined. The second band on the Western blot migrated at 70 kDa, suggesting it most likely represented PDE11A3. This band was most prominent in prostate, much weaker in skeletal muscle, corpus cavernosum, and testis, and totally absent in bladder. The very faint bands of
50 kDa seen in many tissues are believed to be PDE11A1. The sizes of these major bands recognized by the antibody were in good agreement with the predicted molecular mass of PDE11A splice variants (Fawcett et al. 2000
; Hetman et al. 2000
; Yuasa et al. 2000
). No other prominent bands were detected.
There are some discrepancies between our observation and the data in the literature. Disagreements also exist among the published reports regarding the distribution of the PDE11A splice variants. Yuasa et al. (2000) showed that human PDE11A4 mRNA was abundantly and specifically expressed in prostate. They reported that it was not expressed in skeletal muscle, testis, and 12 other tissues surveyed. The authors also showed that within the same panel of tissues, PDE11A3 mRNA was only weakly expressed in testis and not in skeletal muscle or the other tissues. Also in contrast to our finding, Fawcett et al. (2000)
reported that in Western blots of human prostate, only a 56-kDa protein representing PDE11A1 was detected. They also reported that skeletal muscle had one major band possibly representing PDE11A3 and two minor bands for PDE11A2 and 11A1. Further study with antibodies specific to different splicing variants are needed to clarify the discrepancies.
The IHC study reported here showed that the distribution of PDE11A protein is ubiquitous. Positive signals were found in smooth muscle cells, epithelial cells, and endothelial cells from many tissues. It is well known that alterations of cyclic nucleotide levels in smooth muscle cells modulate multiple physiological processes. A rapid increase of cAMP or cGMP levels decreases the contractile tone of smooth muscle cells (Hofmann et al. 2000). The functional consequence of this has been beautifully demonstrated through the use of PDE5 inhibitors in the treatment of erectile dysfunction (Boolell et al. 1996
). PDE5 is the major cGMP hydrolyzing PDE in human corpus cavernosal smooth muscle cells. In cases when erectile dysfunction is caused by insufficient cGMP buildup, inhibition of PDE5 facilitates cGMP accumulation initiated by nitric oxide activation of soluble guanylyl cyclase. The elevated cGMP activates protein kinase G and eventually triggers the lowering of intracellular calcium concentration and the relaxation of corpus cavernosal smooth muscle. This results in an increase of blood flow into the sinusoids and, thus, a more rigid erection (Burnett et al. 1992
; Eardley 1997
). The same end point could also be achieved via elevation of cAMP levels (Maggi et al. 2000
). In addition to responding to rapid changes in cyclic nucleotide concentrations, smooth muscle cells have also been shown to alter their rate of proliferation when continuously exposed to elevated cAMP or cGMP levels. Chronic elevation of cAMP or cGMP levels inhibits human arterial smooth muscle cell proliferation, though to different extents and via distinct mechanisms (Rybalkin and Bornfeldt 1999
; Rybalkin et al. 2003
). It has also been shown that agents increasing cGMP levels, such as nitric oxide or nitric oxide donors, exhibit antiproliferative effects on human prostatic smooth muscle cells (Guh et al. 1998
). In most smooth muscle cells, PDE1, 2, 3, 4, and 5 have been identified (Torphy et al. 1993
; Wallis et al. 1999
; Maurice et al. 2003
; Qiu et al. 2003
; Rybalkin et al. 2003
). Now PDE11A can be added to the list. Unlike most of the PDEs found in smooth muscle cells, PDE11A hydrolyzes cAMP and cGMP equally well (Fawcett et al. 2000
; Hetman et al. 2000
). Conceivably, PDE11A could be involved in any of the above-mentioned physiological processes via its ability to modulate levels of both types of cyclic nucleotides.
PDEs also modulate a wide range of cellular functions in various epithelial cells. It has been shown that increasing cAMP concentrations via PDE4 inhibition decreases TNF- stimulated granulocytemacrophage colony-stimulating factor (GM-CSF) secretion in a human bronchial epithelial cell line (Sasaki and Manabe 2004
). GM-CSF is an important pro-inflammatory cytokine, and bronchial epithelial cells play critical roles in regulating inflammatory responses in the airway. In colonic epithelial cells, increases in cAMP levels through various PDE inhibitor treatments result in increases in transepithelial chloride secretion (O'Grady et al. 2002
). It has been observed that in the rat intestinal epithelial cell line IEC18, phosphodiesterase inhibitor inhibits cell proliferation through modulation of TGF-
expression (Diab-Assef et al. 2002
). The prominent expression of PDE11A in different types of epithelial cells and its ability to regulate the level and duration of intracellular cAMP indicate it may participate in many of these functions.
Although relatively few studies have investigated the expression and function of PDEs in endothelial cells, PDE15 have been identified in various types of endothelial cells and PDE3 and PDE4 inhibitors have been shown to decrease endothelial cell proliferation and the expression of adhesion molecules (reviewed in Maurice et al. 2003). Here we show that PDE11A is expressed in the endothelial cells of most of the tissues examined. Its role in endothelium function awaits further study.
It is well recognized that high levels of cAMP can destroy various types of cancer cells in culture. However, most of the agents that can mimic cAMP action or produce the dramatic increase on cAMP concentrations are highly cytotoxic, precluding their use clinically. In comparison, PDE inhibitors that induce moderate cAMP accumulation also inhibit cancer cell proliferation. For example, elevation of cAMP by nonspecific PDE inhibitors hampers growth in several human prostatic cancer cell lines and induces terminal differentiation in some of the lines (Bang et al. 1994; Goto et al. 1999
). Treatments with specific or nonspecific PDE inhibitors result in growth inhibition in small-cell lung carcinoma (Shafer et al. 1998
), acute promyelocytic leukemia (Guillemin et al. 2002
), and malignant glioma cells (Chen et al. 2002
). Although PDE inhibitors might not kill enough cancer cells when used alone, a nonspecific PDE inhibitor could reduce the effective doses of other anti-cancer drugs, thus maintaining the efficacy and decreasing the toxicity (Hirsh et al. 2004
). Other and newer PDE inhibitors could provide better specificity and efficacy and less side effects in cancer therapy. As PDE11A is expressed abundantly in multiple human carcinomas, it could be a potential target.
In summary, we have studied the tissue distribution of PDE11A using IHC and found that it is expressed in smooth muscle cells, epithelial cells, endothelial cells, and several other types of cells in most of the tissues surveyed. Our results suggest that PDE11A could be involved in multiple physiological processes in various tissues through their ability to modulate cAMP and cGMP levels. A PDE11A-specific inhibitor could greatly facilitate the elucidation of its functional roles.
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Acknowledgments |
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Footnotes |
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Received for publication January 18, 2005; accepted January 24, 2005
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