8-Cl-Adenosine enhances 1,25-dihydroxyvitamin D3-induced growth inhibition without affecting 1,25-dihydroxyvitamin D3-stimulated differentiation of primary mouse epidermal keratinocytes

Background Epidermal keratinocytes continuously proliferate and differentiate to form the mechanical and water permeability barrier that makes terrestrial life possible. In certain skin diseases, these processes become dysregulated, resulting in abnormal barrier formation. In particular, skin diseases such as psoriasis, actinic keratosis and basal and squamous cell carcinomas are characterized by hyperproliferation and aberrant or absent differentiation of epidermal keratinocytes. We previously demonstrated that 8-Cl-adenosine (8-Cl-Ado) can induce keratinocyte growth arrest without inducing differentiation. Results To determine if this agent might be useful in treating hyperproliferative skin disorders, we investigated whether 8-Cl-Ado could enhance the ability of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], a known keratinocyte differentiating agent and a clinical treatment for psoriasis, to inhibit keratinocyte growth. We found that low concentrations of 8-Cl-Ado and 1,25(OH)2D3 appeared to act additively to reduce proliferation of primary mouse epidermal keratinocytes. However, another agent (transforming growth factor-beta) that triggers growth arrest without inducing differentiation also coincidentally inhibits differentiation elicited by other agents; inhibition of differentiation is suboptimal for treating skin disorders, as differentiation is often already reduced. Thus, we determined whether 8-Cl-Ado also decreased keratinocyte differentiation induced by 1,25(OH)2D3, as measured using the early and late differentiation markers, keratin 1 protein levels and transglutaminase activity, respectively. 8-Cl-Ado did not affect 1,25(OH)2D3-stimulated keratin 1 protein expression or transglutaminase activity. Conclusions Our results suggest that 8-Cl-Ado might be useful in combination with differentiating agents for the treatment of hyperproliferative disorders of the skin.


Background
The epidermis of the skin serves as a mechanical and water permeability barrier essential for terrestrial life (reviewed in [1]) and is composed primarily of epidermal keratinocytes. These keratinocytes stratify to form several layers. The deepest layer, the stratum basalis or basal layer comprises proliferating cells that continuously divide to regenerate cells lost to the environment. As the cells migrate upward into the first differentiated layer, the stratum spinousum or spinous layer, they cease proliferating and begin to express the intermediate filament proteins, the mature keratins 1 and 10. This early differentiation is followed by a late differentiation program in the stratum granulosum or granular layer, which is marked by the expression of other structural proteins, such as filaggrin and loricrin, and by increased activity of the enzyme, transglutaminase, which forms highly durable γ-glutamylε-lysyl bonds to cross-link the proteins into a tough and resistant shell underneath the plasma membrane. At the boundary of the granular layer and the outermost stratum corneum, or cornified layer, the keratinocytes terminally differentiate, degrading their nuclei and other organelles and releasing lamellar bodies, the lipid contents of which form a water-impermeant barrier. The squames, the flattened remnants of the keratinocytes, and the lipids from the lamellar bodies form a sort of brick and mortar, to prevent water loss, microbial invasion and/or other mechanical insults (reviewed in [2][3][4]).
Previously, we demonstrated that 8-Cl-Ado arrests the growth of primary mouse epidermal keratinocytes without triggering differentiation [29]. Thus, 8-Cl-Ado functions in an analogous fashion to transforming growth factor-β (TGF-β), which also triggers growth arrest, but not differentiation,, of keratinocytes (reviewed in [30]). In contrast with a polypeptide such as TGF-β, 8-Cl-Ado, as a small molecule rather than a protein, could potentially be taken orally or applied topically to skin. Thus, 8-Cl-Ado may represent a novel therapy for treatment of skin disorders, such as psoriasis, actinic keratoses and basal and squamous cell carcinomas, characterized by hyperproliferation of keratinocytes. One potential problem, however, is that TGF-β also inhibits the expression of differentiation markers elicited by other differentiating agents [31]. Since another characteristic typical of hyperproliferative skin diseases such as psoriasis is impaired differentiation [32], a therapy that inhibits both proliferation and differentiation would be less than ideal.
To determine whether 8-Cl-Ado, as a potent keratinocyte growth arrestor, could potentially be used to treat hyperproliferative skin diseases in combination with a current treatment, we investigated the effect of 8-Cl-Ado on 1,25(OH) 2 D 3 -induced inhibition of keratinocyte proliferation and stimulation of keratinocyte differentiation. We found that low concentrations of 8-Cl-Ado acted additively with 1,25(OH) 2 D 3 to inhibit DNA synthesis, without affecting the ability of 1,25(OH) 2 D 3 to enhance keratin 1 expression, a marker of early differentiation, or transglutaminase activity, a marker of late differentiation. Thus, our results suggest that a combination therapy with 1,25(OH) 2 D 3 and 8-Cl-Ado could potentially be an effective treatment for hyperproliferative skin disorders including psoriasis, actinic keratosis and non-melanoma skin cancers.

Results and discussion
To determine if 8-Cl-Ado could function with the growth inhibiting agent 1,25(OH) 2 D 3 to enhance its antiproliferative effect, we incubated primary epidermal keratinocytes for 24 hours with various concentrations of 8-Cl-Ado in the presence and absence of low concentrations of 1,25(OH) 2 D 3 prior to assessing effects on de novo DNA synthesis as measured by [ 3 H]thymidine incorporation into DNA. As shown in Figure 1A  incorporation that was significantly greater than that elicited by either agent alone. Indeed, the inhibition elicited by 10 µM 8-Cl-Ado and 10 nM 1,25(OH) 2 D 3 was comparable to the inhibition produced by 100 nM 1,25(OH) 2 D 3 alone (compare Figures 1B and 2). Thus, our results suggest that not only does 8-Cl-Ado not prevent the growth inhibitory action of 1,25(OH) 2 D 3 , but, in fact, the two agents seem to act in an additive fashion to more effectively inhibit keratinocyte proliferation.
TGF-β, another agent that, like 8-Cl-Ado, induces growth arrest but not differentiation of keratinocytes ( [31] and reviewed in [30]), can inhibit the ability of differentiating agents to elicit keratinocyte differentiation [31]. However, for an agent to have therapeutic potential as a treatment   Figure 2 to produce a greater growth inhibition than either agent alone. As illustrated in Figure 3, 10 µM 8-Cl-Ado alone had little or no effect on transglutaminase activity, as reported previously [29]. On the other hand, 10 nM 1,25(OH) 2 D 3 significantly elevated transglutaminase activity by approximately 75%. The combination of 8-Cl-Ado and 1,25(OH) 2 D 3 was not significantly different from 1,25(OH) 2 D 3 alone, with a significant approximate 60% increase relative to the control value. Thus, our results indicate that 8-Cl-Ado did not prevent the differentiative effect of 1,25(OH) 2 D 3 , suggesting that these two agents might be combined to treat keratinocyte hyperproliferative disorders, such as psoriasis.  Transglutaminase activity is a marker of late keratinocyte differentiation. We also examined the effect of 8-Cl-Ado on a marker of early keratinocyte differentiation, namely keratin 1 protein expression, using an even higher concentration of 8-Cl-Ado (25 µM). Western analysis demonstrated that 1,25(OH) 2 D 3 induced an approximate 45% increase in keratin 1 protein levels with the combination of 1,25(OH) 2 D 3 and 8-Cl-Ado producing a comparable 46% increase (Figure 4). Thus, early differentiation in response to 1,25(OH) 2 D 3 also was not affected by 8-Cl-Ado. Interestingly, however, in contrast to previous results [29], in these experiments 8-Cl-Ado alone elicited a small but significant increase in keratin 1 protein expression (32%). The reason for this disparity is unclear but may result from differences in the lot of anti-keratin 1 antibody used in the western analysis and/or the increased sensitivity of the method used for detecting and quantifying immunoreactive protein in this work.

8-Cl-Ado Has No Effect on 1,25(OH) 2 D 3 -Stimulated Transglutaminase Activity
Most current treatments for psoriasis suffer from one or more disadvantages including lack of efficacy, contraindications due to deleterious side effects and/or aesthetic deficiencies ( [35] and reviewed in [36]). Indeed, monotherapies tend to be less efficacious than combination therapies with two or more agents used concurrently, sequentially or in a rotational fashion (reviewed in [36]). Treatment with 1,25(OH) 2 D 3 and its analogs has proven successful, although the possibility of toxicity as the result of 1,25(OH) 2 D 3 's ability to affect calcium metabolism has led to the search for topically effective analogs with little or no effect on serum calcium levels (reviewed in [32]). If the amount of 1,25(OH) 2 D 3 (or its analog) required for treatment could be reduced, this decrease in dosage would presumably minimize systemic effects on calcium, which is the primary dose-limiting factor in the use of 1,25(OH) 2 D 3 analogs in the treatment of psoriasis [32]. Thus, our results indicating that 8-Cl-Ado enhances the growth inhibitory effect of 1,25(OH) 2 D 3 , a known keratinocyte differentiating agent and possible treatment for psoriasis [32], suggests the potential for combination therapy. Moreover, the fact that 8-Cl-Ado does not interfere with the promotion of differentiation by 1,25(OH) 2 D 3 further supports the possible combined use of these two agents for treatment of hyperproliferative skin disorders.
Several lines of evidence suggest that 8-Cl-Ado is not simply acting through cyototoxicity to inhibit keratinocyte growth. First, we have previously shown that 8-Cl-Ado growth arrests keratinocytes in the G 0 /G 1 phase of the cell cycle with no increase in the sub-G 0 /G 1 (apoptotic) population of cells [29]. Second, we also showed that the effect of 8-Cl-Ado to inhibit proliferation is reversible in that washout of the compound returned DNA synthesis essentially to basal (untreated) levels [29]. Finally, in this report we demonstrate that 8-Cl-Ado did not inhibit the 1,25(OH) 2 D 3 -stimulated increase in transglutaminase activity (Figure 3) or keratin 1 protein expression ( Figure  4). Together, these results indicate that 8-Cl-Ado is acting in a specific manner to decrease keratinocyte proliferation.
Nevertheless, the mechanism by which 8-Cl-Ado exerts its growth inhibitory effects in keratinocytes is not clear. Our previous results indicate that 8-Cl-Ado must enter the cells to trigger growth arrest, since inhibiting uptake with an adenosine transporter, NBTI, prevented the arrest in the G 0 /G 1 phase of the cell cycle [29]. We also reported in a prior publication that 8-Cl-Ado induced the expression of the cyclin-dependent kinase inhibitor, p21 [29], which is known to contribute to growth arrest in keratinocytes and other cell types ( [37] and reviewed in [30]). However, other investigators have reported 8-Cl-Ado-mediated inhibitory effects on RNA synthesis and the levels of cellular ATP [16]. Clearly, further research is necessary to define the pathways used by 8-Cl-Ado to regulate keratinocyte proliferation.

Conclusions
In summary, our data show that 8-Cl-Ado functions with the keratinocyte-differentiating agent 1,25(OH) 2 D 3 to inhibit keratinocyte proliferation without altering the ability of 1,25(OH) 2 D 3 to induce differentiation. Thus, our results support the possibility of using 8-Cl-Ado alone or in combination with differentiating agents such as 1,25(OH) 2 D 3 or its analogs to treat hyperproliferative keratinocyte disorders including psoriasis.

Materials
Tissue culture reagents were obtained from standard suppliers as indicated in a previous publication [29]

Keratinocyte culture
Primary cultures of mouse epidermal keratinocytes were prepared from neonatal ICR CD-1 mice and cultivated in a 25 µM calcium-containing serum-free keratinocyte medium as in [29].

Measurement of DNA synthesis
For measurement of [ 3 H]thymidine incorporation into DNA, as in [29], near-confluent cultures were refed with SFKM containing various concentrations of 8-Cl-Ado with or without different concentrations of 1,25(OH) 2 D 3 . After 24 hours, cells were labeled with 1 µCi/ml [ 3 H]thymidine for an additional hour in the continued presence of 8-Cl-Ado and/or 1,25(OH) 2 D 3 . Cultures were washed twice with phosphate-buffered saline without calcium or magnesium (PBS -) and macromolecules were precipitated using ice-cold 5% trichloroacetic acid (TCA). After additional washing with 5% TCA and distilled water, cells were solubilized in 0.3 M NaOH, and the amount of [ 3 H]thymidine incorporated into DNA was determined by liquid scintillation counting.

Measurement of transglutaminase activity
Transglutaminase activity was assessed essentially as described in [33]. Briefly, near-confluent keratinocytes were incubated for 24 hours with the indicated agents in SFKM. The cells were scraped into homogenization buffer (0.1 M Tris-acetate, pH 7.8, 2 µg/ml aprotinin, 2 µM leupeptin, 1 µM pepstatin A, 0.2 mM EDTA and 0.2 mM PMSF), collected by centrifugation and subjected to one freeze-thaw cycle prior to disruption by sonication. Aliquots of the homogenate were removed for determination of protein content and transglutaminase activity. Transglutaminase activity was measured as the [ 3 H]putrescine radioactivity incorporated into casein after an overnight incubation at 37°C. Casein was precipitated with TCA, collected onto glass fiber filters and counted by liquid scintillation spectrometry. The cellular protein content of the samples was determined using the Bio-Rad DC protein assay system (Bio-Rad, Hercules, CA), with BSA as standard, and transglutaminase activity was expressed as cpm/µg protein.

Western analysis of keratin 1 protein levels
Keratinocytes were treated and solubilized in sample buffer (31.2 mM Tris, pH 6.8, 1% SDS, 12.5% glycerol). Equal sample volumes were separated by SDS polyacrylamide gel electrophoresis on an 8% gel and transferred to Immobilon PVDF membrane (Millipore, Billerica, MA). Membranes were blocked with Odyssey blocking buffer (Licor Biosciences, Lincoln, NE), probed with a rabbit polyclonal anti-keratin 1 antibody (Covance, Princeton, NJ) and a mouse monoclonal anti-actin antibody (Sigma, St. Loius, MO). Immunoreactive proteins were visualized with IRDye800-coupled donkey anti-rabbit IgG (Rockland Immunochemicals, Gilbertsville, PA) or IR Alexa Fluor 680-coupled goat anti-mouse IgG (Molecular Probes, Eugene, OR) on a Licor Odyssey Infrared Imaging System. Keratin-1 protein levels were corrected for background and normalized using background-corrected actin levels.

Statistical analysis
Significance of differences was determined with the computer program InStat (Graphpad Software, San Diego, CA) using ANOVA with a Student-Newman-Keuls posthoc test.