The Del1 deposition domain can immobilize 3α-hydroxysteroid dehydrogenase in the extracellular matrix without interfering with enzymatic activity
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Developing methods that result in targeting of therapeutic molecules in gene therapies to target tissues has importance, as targeting can increase efficacy and decrease off target-side-effects. Work from my laboratory previously showed that the extracellular matrix protein Del1 is organized in the extracellular matrix (ECM) via the Del1 deposition domain (DDD). In this work, a fusion protein with DDD was made to assay the ability to immobilize an enzyme without disrupting enzymatic function. A prostatic cancer-derived cell line LNCap that grows in an androgen-dependent manner was used with 3α-hydroxysteroid dehydrogenase (3 αHD), which catalyzes dihydrotestosterone (DHT). Plasmids encoding a 3αHD:DDD fusion were generated and transfected into cultured cells. The effects of 3αHD immobilized in the ECM by the DDD were evaluated by monitoring growth of LNCap cells and DHT concentrations. It was demonstrated that the DDD could immobilize an enzyme in the ECM without interfering with function.
KeywordsDel1 Extracellular matrix Immobilization Gene therapy
Mouse Del1 is an ECM protein secreted by embryonic endothelial cells and hypertrophic chondrocytes . Del1 consists of five domains: three epidermal growth factor (EGF) repeat domains (E1, E2, E3) and two Discoidin domains (C1, C2). Work from my laboratory recently showed that the C-termini of the C1 domains are essential for organization of Del1 into the ECM and that both the E3 repeat domain and the N-terminus of the C1 domain play supportive roles in organization into the ECM . We termed this portion of the C1 domain the Del1 deposition domain (DDD). Fusion proteins that include the DDD and an alkaline phosphatase protein as a marker accumulate in the ECM without interfering with enzyme activity of the alkaline phosphatase.
In some therapeutic treatments, localization of bioactive molecules specifically to the target tissue(s) increases the concentration of bioactive molecules in the target cells. This results in higher efficiency of treatment and because the concentration of the bioactive molecules in non-target tissues is minimized, this can also result in a lower incidence of treatment side effects. The purpose of this study is to ask if addition of DDD to an enzyme can result in localization of that enzyme to the ECM without affecting activity of the enzyme. Because the space between a cell and the ECM is not a simple vacancy, it is important to ask if a fusion protein derived from an enzyme is functional in the microenvironment of the ECM.
Cell culture and evaluation of cell growth
LNCap (CRL-1740) and Cos-7 (CRL-1651) cells were purchased from ATCC and grown in α-minimum essential medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen) at 37°C in an atmosphere containing 5% CO2. To evaluate dependency of cell growth on DHT, 50% confluent LNCap cells were prepared in a 24 well plate with 300 μl of a serum free medium, VP-SFM (Invitrogen) and different concentrations of DHT (Wako, Osaka, Japan) were added. After 4 days of culture, 30 μl of WST-1 (Takara, Outs, Japan) were added to the medium, incubated for 1 hour, and the absorbance at 405 nm was measured. The experiment was repeated three times and representative data are shown.
First, a synthetic oligonucleotide encoding the signal peptide from Del1 (MKHLVAAWLLVGLSLGVPQFGKGDI) was obtained and cloned into pcDNA3D (Invitrogen) resulting in pcDNA3S. Next, a cDNA fragment encoding the human 3αHD gene (AB178898) was amplified by reverse transcriptase-PCR (RT-PCR) using the forward primer, 5′-AAGAATTCATGAACTCCAAATGTCATTGTGTCAT and the reverse primer 5′-AAGATATCGTATTCATCCAAAAATGGCCAATTAG. The amplified fragment was cloned into the 3′ end of the signal peptide, resulting in p3αHD (Fig. 1a). Finally, a fragment encoding the DDD (amino acids 122–316 of Del1) was amplified with the forward primer 5′-AAGATATCTGTGAAGCTGAGCCTTGCAGAAAT and the reverse primer 5′-AACTCGAGCATGTCCATGTTGAGTGTTCTGAA. This was cloned into the 3′ end of the 3αHD gene, resulting in the plasmid p3αHD:DDD (Fig. 1a). The recombinant proteins expressed by these constructs also had a V5 epitope tag at their C terminal ends.
ECM samples for immunoblotting analysis were prepared according to Hidai et al. . Briefly, Cos-7 cells in six-well plate were transfected with pcDNA3S, p3αHD or p3αHD:DDD using Lipofectamine 2000 (Invitrogen). After 96 h of culture, conditioned medium was harvested. Then, cells were induced to detach by treatment of EDTA and the remaining ECM was collected with a scraper. The conditioned medium, cells, and ECM were fixed with 10% trichloracetic acid (Wako). The protein samples were separated by SDS-PAGE and transferred to a PVDF membrane (ATTO, Tokyo, Japan). The membrane was incubated with anti-V5 antibody (Invitrogen), followed by incubation with a horseradish peroxidase-conjugated secondary antibody, and immunoreactive proteins were detected using the ECL Advance Western Blotting Detection Kit (Amersham, Piscataway, NJ).
Evaluation of the effects of exogenous 3αHD
LNCap cells were plated to 50% confluency in a 24-well dish and transfected with 1 μg of pcDNA3S, p3αHD, or p3αHD:DDD using Lipofectamine2000. To test if an excess of p3αHD was introduced, cell growth was monitored after introduction of various amount of p3αHD. To monitor transfection efficiency, 0.1 μg of cDNA for the LacZ gene was co-transfected with each test construct. Expression of LacZ gene was monitored by measuring β-galactosidase enzyme activity using an assay system (Promega, Madison, WI). Cell number was evaluated with WST-1. The values obtained in samples transected with the control plasmid, pcDNA3S, were normalized to 1. The experiment was repeated three times and representative data are shown.
Assays of conditioned ECM
ECM conditioned with 3αHD was prepared according to Hidai et al. . Briefly, Cos-7 cells were cultured in a 24-well dish and transfected with 1 μg of pcDNA3S, p3αHD, or p3αHD:DDD using Lipofectamine2000. To standardize transfection efficiency, 0.1 μg of LacZ cDNA were co-transfected. Four days later, the cells were washed with phosphate buffered saline (PBS) and incubated in PBS containing 10 mM EDTA and a protease inhibitor cocktail (PIERCE, Rockford, IL) for 12 h at 4ºC. Next, cells were harvested by vigorous pipetting, collected by centrifugation, resuspended in 100 μl of tris buffered saline (TBS), and lysed by sonication. β-Galactosidase activity of the cell lysates was then assayed. Next, 1 × 104 LNCap cells were plated on the conditioned ECM, cultured for seven days, and evaluated with WST-1. To ask if exogenous DHT could rescue the growth of LNCap cells, various amounts of DHT (Wako) were added to the medium. The values obtained for samples transfected with the control plasmid, pcDNA3S, were normalized to 1. The experiment was repeated three times and representative data are shown.
Measurement of dihydrotestosterone levels
ECM conditioned with 3αHD was prepared as described above. Serum obtained from a male volunteer as approved by Nihon University ethics committee was incubated with conditioned ECM in the presence of a protease inhibitor cocktail (PIERCE) for 96 h. The concentration of dihydrotestosterone in the serum was measured using the Dihydrotestosterone ELISA kit (IBL, Hamburg, Germany) according to the manufacture’s protocol. The values obtained for samples transfected with the control plasmid, pcDNA3S, were normalized to 1. The experiment was repeated three times and representative data are shown.
Results were expressed as mean ± SEM. Dunn’s tests or a Wilcoxon’s test were performed, and statistical significance was set at P < 0.01.
Results and discussion
To determine if there is a dose-dependent relationship between cell growth and the amount of p3αHD DNA transfected into cells, the effect of transfection with different amounts of p3αHD DNA was tested (Fig. 3b). The effect on cell growth was dependent on the amount of p3αHD added, suggesting that suppression of cell growth was not due to experimental procedures but instead, is the result of 3αHD-specific effects.
On explanation for the effect observed in this work is that the presence of 3αHD in the ECM decreases the concentration of DHT in serum. To test this, serum was incubated with conditioned ECM and the concentration of DHT was measured in treated and untreated samples (Fig. 4b). After 96 h, the concentration of DHT in serum incubated with ECM conditioned with p3αHD:DDD had significantly decreased.
To confirm that the p3αHD:DDD suppresses the growth of LNCap cells via a decrease of DHT in medium, the ability of exogenously added DHT to reverse the effect was assayed (Fig. 4c). Administration of exogenous DHT was able to restore growth of LNCap cells even in the presence of p3αHD:DDD.
In a previous study, it was found that alkaline phosphatase protein was efficiently deposited in the ECM in vitro and in vivo . The present study suggests that DDD can fix enzymes in the ECM in a functional state, suggesting some biomedical applications for DDD fusion proteins. In cytotherapy, for example, cells could be pre-engineered with a cDNA encoding a specific DDD fusion protein. Because DDD has been reported to be effective for targeting to ECMs from various kinds of cells, one can predict that cell choice may be dictated solely according to need and that the ECM conditioned by the cells can then serve as a source of active enzyme.
Similarly, a system involving a DDD fusion protein under the control of a tissue-specific promoter could efficiently localize an exogenous protein useful in gene therapy and thus presumably, target the effects of that enzyme to a specific cell type and/or location. This has promise for generating regions of high local concentration while at the same time, resulting in only a low systemic concentration of the therapeutic proteins by virtue of their deposition in ECM. To apply the idea further, one can imagine that chemicals capable of being activated by an enzyme may then by activated in a limited region, such as in a specific target tissue or cell type, via expression of an ECM-anchored version of the activating enzyme.
In addition to enzymes, some ligands, such as the FAS ligand, might be used as partners of DDD and the system adapted to other applications . That is, the presence of a cytotoxic ligand may limit the number of cells that can produce the ligand but if that ligand is in the ECM via anchoring by DDD, the ligand may remain active and accessible in the ECM even after death of the cell that initially expressed it. If this type of fusion between DDD and cytotoxic ligands results in a protein that is fixed in the ECM and significantly active, then one can imagine development of additional applications for the system.
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