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The Journal of Clinical Endocrinology & Metabolism Vol. 88, No. 12 5704-5709
Copyright © 2003 by The Endocrine Society

Increased Expression of the 25-Hydroxyvitamin D3-1{alpha}-Hydroxylase Gene in Alveolar Macrophages of Patients with Lung Cancer

Koushi Yokomura, Takafumi Suda, Shigekazu Sasaki, Naoki Inui, Kingo Chida and Hirotoshi Nakamura

Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu 431-3192, Japan

Address all correspondence and requests for reprints to: Hirotoshi Nakamura, Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, 1-20-1 Handayama, Hamamatsu 431-3192, Japan. E-mail: hirotosh{at}hama-med.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
25-Hydroxyvitamin D3-1{alpha}-hydroxylase (1{alpha}-hydroxylase) plays a central role in calcium metabolism by synthesizing the active hormone 1{alpha},25-dihydroxyvitamin D3 in the kidney. Its increased expression in the extrarenal tissues has been found in alveolar macrophages in sarcoidosis but not in any other pathological conditions. We found that 1{alpha}-hydroxylase-mRNA in alveolar macrophages measured by semiquantitative RT-PCR was 2-fold greater in patients with lung cancer than in control subjects (0.61 ± 0.20 vs. 0.34 ± 0.11, respectively; P < 0.0001). When the clinical stages of lung cancer were divided into early (stage IA–IIIA) and advanced (stage IIIB and IV) and the expression of 1{alpha}-hydroxylase gene was compared among the control, early, and advanced groups, the advanced group showed the highest expression, followed by the early group, then the control group (0.34 ± 0.11, 0.52 ± 0.11, and 0.69 ± 0.23 for control, early, and advanced groups, respectively; P < 0.0001). The 1{alpha}-hydroxylase-mRNA level was well correlated with serum 1{alpha},25-dihydroxylase D3 concentration and the 1{alpha},25-dihydroxylase D3 to 25-hydroxyvitamin D3 ratio, but none of the findings related to calcium metabolism among the patients with lung cancer. Increased local production of 1{alpha},25-dihydroxyvitamin D3 may be associated with the pathological conditions, such as immunosuppression, in lung cancer.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
25-HYDROXYVITAMIN D3-1{alpha}-HYDROXYLASE (1{alpha}-hydroxylase) is the mitochondrial cytochrome P450 enzyme that converts 25-hydroxyvitamin D3 [25-(OH) D3] to 1{alpha},25-dihydroxyvitamin D3 [1,25-(OH)2 D3] (1, 2, 3, 4). The enzyme activity of 1{alpha}-hydroxylase is the key determinant to the synthesis of the active hormone 1,25-(OH)2 D3, which plays an important role in not only calcium metabolism but also cell differentiation and proliferation (2, 3, 4). Actually, 1,25-(OH)2 D3 has been shown to have potent immunosuppressive properties (5). Under normal circumstances, the hydroxylation of 25-(OH) D3 is exclusively conducted in the proximal renal tubule. Certain pathological conditions, such as sarcoidosis and other granulomatous diseases, cause extrarenal expression of this enzyme, resulting in possible hypercalcemia and hypercalciuria (6, 7, 8). Several investigators have recently isolated rat, mouse, and human 1{alpha}-hydroxylase cDNA (9, 10, 11, 12, 13). Using the semiquantitative RT-PCR technique, we identified, for the first time, the expression of the 1{alpha}-hydroxylase gene in human alveolar macrophages (AMs) (14). We also demonstrated that the 1{alpha}-hydroxylase-mRNA level in AMs obtained from patients with sarcoidosis was significantly higher than the level obtained from the control patients with other pulmonary diseases (14). The 1{alpha}-hydroxylase-mRNA level was well correlated with the disease activity of sarcoidosis, serum ionized calcium, and the ratio of 1,25-(OH)2 D3 to 25-(OH) D3. The function of increased 1{alpha}-hydroxylase in sarcoidosis is uncertain, but our findings raised the possibility that 1,25-(OH)2 D3 might provide a compensatory mechanism to inhibit the inflammatory process, suppressing the activated helper T cell proliferation and cytokine production in sarcoid granulomas (15, 16, 17).

In the present study, we found the significantly increased expression of 1{alpha}-hydroxylase in AMs of patients with lung cancer. The level of 1{alpha}-hydroxylase-mRNA was well correlated to serum 1,25-(OH)2 D3 and the ratio of serum 1,25-(OH)2 D3 to 25-(OH) D3 but not to calcium metabolism. The enzyme expression increased depending on the progress in the clinical stage.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Twenty-two consecutive patients with lung cancer (14 men and eight women, ages 44–83 yr) who underwent bronchoalveolar lavage (BAL) between October 1999 and June 2003 were included in this study. The diagnosis of lung cancer was histologically established on biopsy samples of lung tissues. The clinical features of these patients are shown in Table 1Go. The histological types of lung cancer were adenocarcinoma in 12 patients, small-cell carcinoma in five patients, and squamous cell carcinoma in five patients. Their clinical stages were stage IA in five patients, stage IB in one patient, stage IIA in one patient, stage IIIA in two patients, stage IIIB in 10 patients, and stage IV in three patients. BAL was performed before any medical treatment in 19 patients, but patients no. 14, 15, and 20 had received chemotherapy with or without radiotherapy before BAL. None of the patients had received medications that might affect calcium metabolism. There were nine smokers and 13 nonsmokers. For controls, 13 patients with other pulmonary disorders (five patients with idiopathic interstitial pneumonia, four patients with pulmonary emphysema, and four patients with organizing pneumonia) and five normal healthy volunteers were studied. Because no significant difference was observed between the two groups in any experimental measurements, as shown in Results, all subjects were combined in a control group (16 men and two women, ages 30–77 yr, six smokers and 12 nonsmokers). The study protocol was approved by our institutional review board for human research. Informed consent was given by each subject.


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  		TABLE 1. Clinical characteristics of patients with lung cancer

 
BAL procedures, preparation of BAL cells, and purification of AMs

BAL was performed as previously reported (18). Briefly, both the upper and lower respiratory tracts were anesthetized with topically administered lidocaine. A BF-240 Olympus fiberoptic bronchoscope (Olympus Co., Tokyo, Japan) was passed transorally and wedged in a segmental or subsegmental bronchus. Three 50-ml aliquots of prewarmed sterile 0.9% saline were instilled, and the returns were gently aspirated through the side channel of the bronchoscope (19). Bronchoalveolar cells were separated by centrifugation at 500 x g for 10 min at 4 C and washed with PBS. The total cell count was determined using a hemocytometer. The BAL cells were resuspended in the culture medium at a density of 1 x 106 cells/ml. A differential cell count was performed on cytocentrifuged smears stained with Wright-Giemsa. As in the previous study (14), purified AMs were obtained by the method of removing nonadherent cells (20). The cells were plated into dishes coated with fetal calf serum and incubated at 37 C in 5% CO2 for 2 h. All adherent cells were macrophages.

Measurement of serum markers of calcium metabolism

Fasting blood samples were collected at the time of bronchoscopy. The serum calcium level, which was measured by automated methods, was corrected for the serum albumin level. The serum levels of PTH and PTHrP were determined by chemiluminescent immunoassay (normal range, 10–65 ng/l) and immunoradiometric assay (normal range, < 0.6 pmol/l), respectively. The serum ionized calcium level was measured using an ion-selective electrode (normal range, 1.20–1.35 mmol/l) (21). The serum levels of 1,25-(OH)2 D3 (normal range, 26–169 pmol/l) and 25-(OH) D3 (normal range, 25–137 nmol/l) were measured by RIA and competitive protein-binding analysis (22), respectively.

RNA isolation and RT-PCR

Total RNA was extracted from BAL cells using the acid guanidinium thiocyanate-phenol-chloroform technique (23). Two micrograms were used for the first-standard cDNA synthesis with Moloney murine leukemia virus reverse transcriptase (Life Technologies, Rockville, MD) and random hexanucleotide. After terminating the reaction by heating at 70 C for 10 min, the reaction mixture was diluted 5-fold with distilled water.

The level of human 1{alpha}-hydroxylase-mRNA was measured by PCR amplification. PCR was performed in 50 µl reaction mediums containing 5 µl of cDNA solution, 0.2 mM of forward and reverse primers, 0.2 mM of deoxynucleotide triphosphates, and 1.25 U of Pyrobest DNA polymerase (Takara Shuzo Co., Tokyo, Japan). After 3 min of initial denaturation at 96 C, amplification was performed in a DNA Thermal Cycler (Perkin-Elmer, Norwalk, CT) for 42 cycles with 30-sec denaturing at 96 C, 40-sec annealing at 60 C, and 1-min extension at 72 C. We designed the sequences of the primers spanning an intron of each genome, 5'-GTGTCCACGCTGTTGACCATG-3' and 5'-GAATTTCCAGGTACCACAGG-3'. When semiquantitative RT-PCR was performed, PCR amplification was carried out for 30–42 cycles for 1{alpha}-hydroxylase and for 28–38 cycles for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the control for gene expression. The PCR products were separated on 1% agarose gel by electrophoresis, stained with ethidium bromide, and visualized. We confirmed the linearity of the PCR products up to 36 cycles for 1{alpha}-hydroxylase and GAPDH; the values obtained were plotted against the numbers of cycles. Each best-fit straight line was obtained and extrapolated back to zero cycle. The intercepts at the zero cycle were used to determine the relative abundance of both mRNAs, and the value for 1{alpha}-hydroxylase was normalized to GAPDH. The RT-PCR product of 1{alpha}-hydroxylase was confirmed by Southern blot analysis as described previously (14).

Statistical analysis

All values were analyzed with StatView J 4.5-software (SAS Institute, Cary, NC). The BAL cells, serum markers of calcium metabolism, and 1{alpha}-hydroxylase mRNA level were compared among groups by ANOVA, and when ANOVA showed significant difference, Fisher’s protected least significant difference test was performed. P < 0.05 was considered significant. The correlation between relative intensity of the 1{alpha}-hydroxylase mRNA level and markers of calcium metabolism were evaluated using the Pearson’s correlation coefficient. All continuous values are expressed as means ± SD.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Expression of the 1{alpha}-hydroxylase gene in AMs obtained from patients with lung cancer

Between the patients with lung cancer and the control subjects, there were no significant difference in the total counts of BAL cells obtained and the percentages of macrophages and lymphocytes (Table 2Go).


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  		TABLE 2. BAL and laboratory findings of patients with lung cancer and controls

 
The 1{alpha}-hydroxylase-mRNA was detected in AMs obtained from every subject. The relative intensity of its level measured by semiquantitative RT-PCR is shown in Fig. 1Go. No difference was found between five normal healthy volunteers and 13 patients with other pulmonary diseases; therefore, we combined them in a control group. The expression of 1{alpha}-hydroxylase mRNA in patients with lung cancer was approximately 2-fold greater than that in the controls (0.61 ± 0.20 vs. 0.34 ± 0.11, P < 0.0001).



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  		FIG. 1. Difference in the expression of 1{alpha}-hydroxylase mRNA in AMs between the patients with lung cancer and controls. In the controls, shaded circles represent normal healthy volunteers, whereas open circles represent patients with other pulmonary diseases. There was no difference in the expression of 1{alpha}-hydroxylase mRNA between the normal healthy volunteers and control patients with other pulmonary diseases.

 
Correlation of the 1{alpha}-hydroxylase-mRNA level and the findings concerned with lung cancer

When we compared the 1{alpha}-hydroxylase-mRNA expression level among the clinical stages of lung cancer, it was difficult to make a clear conclusion because the number of the patients in each group was small, although a tendency might be seen that the enzyme expression increased with advancing clinical stage (Fig. 2AGo). Therefore, we divided the clinical stage into early and advanced stage, according to the clinical classification for operability (24). Early lung cancer includes stage IA–IIIA, whereas advanced lung cancer includes stage IIIB and IV. When the expression of 1{alpha}-hydroxylase gene was compared among control, early, and advanced groups, the advanced group had the highest mRNA levels, followed by the early group, and then the control group (Fig. 2BGo). No difference was observed among the pathological types of lung cancer (Fig. 2CGo), or between genders, smoking habits, or prior treatments (data not shown).



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  		FIG. 2. Comparison of the expression of the 1{alpha}-hydroxylase gene among the patients with lung cancer between their clinical stages (A and B) and their pathological types (C). The early stage of lung cancer includes stages IA–IIIA, and the advanced stage of lung cancer includes stages IIIB and IV. In the controls, shaded circles represent normal healthy volunteers, whereas open circles represent patients with other pulmonary diseases. Horizontal lines with figures represent mean values for each group of circles.

 
Relationship between the 1{alpha}-hydroxylase-mRNA level and calcium metabolism

The 1{alpha}-hydroxylase-mRNA level showed a significant correlation with the serum 1,25-(OH)2 D3 concentration (P = 0.03, Fig. 3AGo) and with the ratio of 1,25-(OH)2 D3 to 25-(OH) D3 (P = 0.0008, Fig. 3BGo) among the patients with lung cancer. The correlation became much higher when analyzed in all 40 patients, including patients with lung cancer, control patients with other pulmonary diseases, and healthy volunteers (Fig. 3Go, A and B). As shown in Table 2Go, none of the findings related to calcium metabolism, such as serum calcium, ionized calcium, PTH, PTHrP, 25-(OH) D3 and 1,25-(OH)2 D3, were significantly different between the lung cancer group and the controls. However, the ratio of 1,25-(OH)2 D3 to 25-(OH) D3 was significantly higher in the patients with lung cancer than control subjects, suggesting that the enzyme was functional. When compared among control, early, and advanced groups, the serum 1,25-(OH)2 D3 level tended to be higher, although not significant, in the advanced group; and the ratio of 1,25-(OH)2 D3 to 25-(OH) D3 was significantly higher in the advanced lung cancer group than other groups (Fig. 4BGo).



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  		FIG. 3. Correlation of the expression of the 1{alpha}-hydroxylase gene and the serum concentration of 1,25-(OH)2 D3 (A) and the ratio of 1,25-(OH)2 D3 to 25-(OH) D3 (B). The closed circles, shaded circles, and open circles represent patients with lung cancer, normal healthy subjects, and control patients with other pulmonary diseases, respectively. The solid lines indicate the regression lines among the patients with lung cancer only; the broken lines indicate the regression lines for all subjects, including patients with lung cancer and control subjects.

 


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  		FIG. 4. Comparison of the serum concentration of 1,25-(OH)2 D3 (A) and the ratio of 1,25-(OH)2 D3 to 25-hydroxyvitamin D3 (B) among the clinical stages. In the controls, shaded circles represent normal healthy volunteers, whereas open circles represent patients with other pulmonary diseases. There was no difference in the data between normal healthy volunteers and patients with other pulmonary diseases.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the previous study, we demonstrated the presence of 1{alpha}-hydroxylase gene expression in human AMs and observed a significant correlation between the 1{alpha}-hydroxylase gene expression and the disease activity in patients with sarcoidosis (14). In the present study, using the same semiquantitative RT-PCR technique, we found that the expression of 1{alpha}-hydroxylase mRNA in AMs was significantly enhanced in patients with lung cancer. Except for sarcoidosis, there are few reports demonstrating the enhanced expression of 1{alpha}-hydroxylase gene in human extrarenal tissues under pathological conditions.

A significant correlation was obtained between the 1{alpha}-hydroxylase-mRNA level in AMs of patients with lung cancer and their serum 1,25-(OH)2 D3 concentration and the 1,25-(OH)2 D3 to 25-(OH) D3 ratio. This suggests that 1{alpha}-hydroxylase in AMs in patients does actually function to catalyze the synthesis of active vitamin D3 sterol. In contrast to sarcoidosis, however, the increased expression of this enzyme did not correlate with serum total and ionized calcium concentrations. The enhancement of 1{alpha}-hydroxylase might not be adequate to induce an increase in serum calcium because the patients with sarcoidosis in our previous study showed a 6-fold increase in 1{alpha}-hydroxylase-mRNA, whereas the patients with lung cancer in the present study manifested only twice as much of the enzyme level as the controls.

It is of interest that the 1{alpha}-hydroxylase gene expression tended to be higher in advanced stages of lung cancer. When we classified the patients based on their clinical stages from IA–IV, we did not obtain a significant difference in the 1{alpha}-hydroxylase-mRNA level among the groups because of the insufficient number of patients. The patients were, therefore, divided into two groups, early and advanced; the early group included stages IA–IIIA, and the advanced group included stages IIIB and IV. The basis of this classification is that patients with non-small-cell lung cancer with clinical stages IA–IIIA are usually considered to be operable, whereas patients with stages IIIB and IV are not. The mRNA level of 1{alpha}-hydroxylase was the highest in the advanced group and lowest in the control group (Fig. 2BGo). In addition, the advanced group showed a significantly higher level of the 1,25-(OH)2 D3 to 25-(OH) D3 ratio and a tendency to have a higher 1,25-(OH)2 D3 concentration than the control group (Fig. 4Go).

Although 1{alpha}-hydroxylase is almost exclusively expressed in the kidney under normal circumstances (2, 3, 4, 25), the presence of this enzyme in some extrarenal cells, such as macrophages and keratinocytes, has been suggested under some pathological conditions by the assay of the enzyme activity (2, 4, 25). After cloning of the 1{alpha}-hydroxylase cDNA, the detection of the extrarenal expression of this enzyme at the mRNA and protein levels has become possible. We demonstrated 1{alpha}-hydroxylase-mRNA in human AMs for the first time (14). Zehnder et al. (26) synthesized a specific antibody against an antigenic region of the mouse 1{alpha}-hydroxylase amino acid sequence and examined the extrarenal expression of this enzyme. Their immunohistochemical study confirmed the discrete expression of 1{alpha}-hydroxylase in several human tissues including skin (basal keratinocytes), lymph nodes (granulomata), colon (epithelial cells and parasympathetic ganglia), pancreas (islets), adrenal medulla, brain (cerebellum and cerebral cortex), and placenta (decidual and trophoblastic cells). Thus, the expression of 1{alpha}-hydroxylase is more widespread than previously suggested, but the function of this enzyme in these extrarenal tissues is unknown at present.

It may be intriguing to consider the functional relevance of 1{alpha}-hydroxylase enhancement in lung cancer. Augmented enzyme expression did not relate to the serum calcium level but did relate to the increased synthesis of circulating 1,25-(OH)2 D3, which probably reflected the increased local production induced by the enhanced 1{alpha}-hydroxylase activity in AMs. It was reported that 1,25-(OH)2 D3 plays immunomodulatory roles in a variety of immune responses. In vitro studies have revealed that 1,25-(OH)2 D3 inhibits activated T cell functions, resulting in suppression of cytokine synthesis and antibody-stimulating activities (2, 3, 4, 5, 27). 1,25-(OH)2 D3 has also been demonstrated to inhibit natural killer cell activity at a physiological concentration (28) and suppress the proliferation and maturation of antigen presentation cells (29, 30). Moreover, administration of 1,25-(OH)2 D3 in vivo diminished the TNF{alpha} and interferon (IFN)-{gamma} production by Th1 cells, resulting in prevention of experimental autoimmune encephalomyelitis (31). Vitamin D analogs have been effectively used to prolong transplant survival in animal models of transplantation (32, 33). These findings suggest that 1,25-(OH)2 D3 is a potent immunosuppressive agent and raise the possibility that the locally synthesized 1,25-(OH)2 D3 in AMs function for immunosuppression in lung cancer. It was reported that patients with lung cancer exhibit dysfunction of the immune system, such as the impairment of tumor-specific immune response during the initial stage of tumor growth and generalized immunodeficiency during the late stage of tumor development (34, 35). Alterations in T cell functions occur in patients with various cancers including melanoma (36), renal cell carcinoma (37), cervical cancer (38), and others (39). In addition, mice bearing slowly progressive tumors induced by carcinogen demonstrated significantly diminished functions of T cells and natural killer cells and impaired capacity to produce Th1 cytokines (40).

The mechanism of how 1{alpha}-hydroxylase is regulated in AMs of patients with lung cancer remains to be elucidated. It is known that renal 1{alpha}-hydroxylase synthesis is tightly regulated by the concentration of serum calcium, PTH, and 1,25-(OH)2 D3 (2, 3, 4, 25, 41). In contrast, extrarenal synthesis of 1{alpha}-hydroxylase by macrophages does not respond to these regulatory influences, but rather, it is sensitive to immune signals, such as IFN-{gamma}, TNF{alpha}, and lipopolysaccharide (42, 43, 44, 45, 46). In patients with malignancy, overproduction of various cytokines, including IFN-{gamma}, TNF{alpha}, IL-2, and TGF-ß, have been identified either by cancer cells or host immune cells (34, 35). Their production was most evident in patients with advanced stages of cancer. Although we have currently no data indicating augmentation of these cytokines in BAL, it is possible that they may play a role in enhanced expression of 1{alpha}-hydroxylase in AMs.

In conclusion, the present study demonstrated that patients with lung cancer have increased expression of 1{alpha}-hydroxylase in AMs, which correlates with serum 1,25-(OH)2 D3 concentration. It is possible that increased production of 1,25-(OH)2 D3 may be associated with immunosuppression in lung cancer.


   Footnotes
 
Abbreviations: AM, Alveolar macrophage; BAL, bronchoalveolar lavage; {alpha}-hydroxylase, 25-hydroxyvitamin D3-1{alpha}-hydroxylase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN, interferon; 11,25-(OH)2 D3, 1{alpha},25-dihydroxyvitamin D3; 25-(OH) D3, 25-hydroxyvitamin D3.

Received March 27, 2003.

Accepted September 5, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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