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J Am Soc Nephrol 10:796-803, 1999
© 1999 American Society of Nephrology

REGULAR ARTICLES

Parathyroid Hormone-Related Protein (107-139) Stimulates Interleukin-6 Expression in Human Osteoblastic Cells

FERNANDO DE MIGUEL*, PILAR MARTINEZ-FERNANDEZ{dagger}, CARLOS GUILLEN*, ALVARO VALIN*, ANA RODRIGO{dagger}, MARIA EUGENIA MARTINEZ{dagger} and PEDRO ESBRIT*

* Metabolic Research Unit, Fundación Jiménez Díaz, Madrid, Spain.
{dagger} Biochemistry Division, Hospital La Paz, Madrid, Spain.

Correspondence to Dr. Pedro Esbrit, Metabolic Research Unit, Fundación Jiménez Díaz, Avda. Reyes Católicos, 2, 28040 Madrid, Spain. Phone: 54 41 6005440100; Fax: 54 41 0075491100


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abstract. The N-terminal region of both parathyroid hormone (PTH) and PTH-related protein (PTHrP) binds to the same PTH/PTHrP receptor in osteoblasts. However, C-terminal PTHrP (107-139) inhibits growth and various functions of osteoblasts and osteoclasts apparently through PTHrP-specific receptors. PTH (1-34) and PTHrP (1-34) rapidly induce interleukin-6 (IL-6) expression by osteoblasts. The aim of the present study was to assess the effects of PTHrP (107-139) on IL-6 gene expression and secretion by osteoblastic cells from human trabecular bone (hOB). Using reverse transcription followed by PCR, it was found that IL-6 mRNA was twofold maximally increased by either PTHrP (1-34) or PTHrP (107-139), at 10 nM, over basal within 1 to 2 h in hOB cells. This effect of PTHrP (107-139), and that of PTHrP (1-34), were abolished by the transcription inhibitor actinomycin D. Meanwhile, puromycin, a protein synthesis inhibitor, superinduced IL-6 expression in the presence or absence of each PTHrP peptide. Both PTHrP (1-34) and PTHrP (107-139), but not PTHrP (38-64), stimulated IL-6 secretion to the hOB cell-conditioned medium at 24 h, dose dependently. In addition, this maximal stimulatory effect (twofold over basal) was similar with each PTHrP peptide alone, and not additive when added together. PTHrP (107-139) stimulation of mRNA and protein in hOB cells was abolished by bisindolylmaleimide I, a protein kinase C inhibitor, but not by either adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS), or N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89), two protein kinase A inhibitors. These results indicate that C-terminal PTHrP, like its N-terminal domain, induces IL-6 production by human osteoblastic cells. This effect of both PTHrP regions could provide a mechanism to modulate bone turnover.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Parathyroid hormone-related protein (PTHrP) was initially purified, and its gene cloned, from tumors in patients with humoral hypercalcemia of malignancy (1). PTHrP has now been identified in many fetal and adult normal tissues, including bone, and it appears to function as an auto/paracrine-regulating factor in at least some of these tissues (2,3).

Current evidence points to PTHrP as an important regulator of bone development and metabolism. The N-terminal region of PTHrP has partial homology with parathyroid hormone (PTH). Thus, PTHrP, like PTH, stimulates bone resorption through interaction with PTH/PTHrP receptors in osteoblasts (4,5). PTHrP and the latter receptor are present in osteoblastic cells and chondrocytes apparently depending on their differentiation (6,7,8,9). Various in vitro reports are consistent with an inhibitory effect of N-terminal PTHrP on osteoblastic growth and chondrocyte differentiation (10,11,12,13,14). Moreover, recent studies in mice indicate that targeted disruption of the PTHrP gene induces dramatic abnormalities in endochondral bone formation, similar to those observed in homozygous animals lacking the PTH/PTHrP receptor (15,16). Thus, the effects of PTHrP on chondrocyte growth and/or development seem to be mediated mainly by the PTH/PTHrP receptor. Interestingly, the C-terminal region (88-106) of PTHrP is a consensus nucleolar targeting signal motif, which has been shown to be responsible for the delayed apoptosis of PTHrP-transfected chondrocytes in serum-free medium (17). Moreover, PTHrP (107-139), a putative C-terminal PTHrP fragment (18), appears to affect osteoclastic bone resorption, although its specific effects on osteoclasts remain controversial (19,20,21,22,23,24). In addition, the latter peptide has recently been shown to inhibit bone formation in vitro, as well as in adult mouse calvaria and in the trabecular bone of adult ovariectomized rats (12,13,23,24). The effects of PTHrP on the bone microenvironment, however, were mediated not only by its N-terminal PTH-like region, but also by other C-terminal domains that do not interact with the classical PTH/PTHrP receptor.

Interleukin-6 (IL-6) has been shown to be involved in the increased bone resorption in some cases of malignant hypercalcemia (25,26) and Paget's disease (27). In addition, several studies have found that both PTH and IL-6 are implicated in the bone loss associated with estrogen deficiency (28,29,30). In this regard, various in vitro and in vivo studies suggest that IL-6 is a downstream effector of PTH and PTHrP, and other resorptive agents, to increase osteoclast resorptive activity (31,32,33,34,35,36,37,38).

In the present study, we found that PTHrP (107-139), similarly to PTHrP (1-34), stimulates IL-6 gene expression and IL-6 release in osteoblastic cells from human trabecular bone. Moreover, we demonstrated that this effect of PTHrP (107-139) occurs via a protein kinase C (PKC) signaling mechanism.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Dulbecco's modified Eagle's medium and fetal bovine serum (FBS) were supplied by BioWhittaker (Verviers, Belgium). Human PTHrP (1-34) amide [PTHrP (1-34)], human PTHrP (107-139), actinomycin D, and puromycin were obtained from Sigma (St. Louis, MO). Human PTHrP (38-64) amide [PTHrP (38-64)] was from Peninsula Laboratories (Belmont, CA). Bisindolylmaleimide I (BIM), N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89), and phorbol-12-myristate-13-acetate (PMA) were from Calbiochem (San Diego, CA). Adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS) was from Biolog Life Science Institute (Bremen, Germany).

Cell Culture
Explants of human trabecular bone (hOB) were obtained from hip or knee samples, discarded at the time of surgery on 14 osteoarthritic patients, and cultured as described (13). The patients (7 women and 7 men, 39 to 76 yr) had no evidence of bone metabolic disturbances. The bone fragments were placed in culture flasks with Dulbecco's modified Eagle's medium supplemented with 15% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, in 5% CO2 at 37°C. Confluent cell monolayers of these cells (hOB) have previously been characterized and showed osteoblastic features, such as type I collagen and alkaline phosphatase secretion, 1,25(OH)2D3-stimulated osteocalcin secretion, and N-terminal PTHrP-increased protein kinase A activity (13). Experiments were performed in first-passage cultures after 2 to 4 wk. Preliminary observations showed maximal IL-6 expression, in the presence of 15% FBS, within this time period. hOB cells were preincubated for 48 h in serum- and phenol red-free medium containing 10 nM vitamin K, 50 µg/ml ascorbic acid, and 0.1% bovine serum albumin before addition of test agents in the same medium.

RNA Extraction and Reverse Transcription-PCR
IL-6 mRNA levels were assessed by reverse transcription (RT) followed by PCR. Total RNA was extracted from cells using guanidinium thiocyanate-phenol-chloroform extraction (39). Approximately 6 µg of total RNA was isolated from hOB cells grown in one 35-mm dish. Total RNA aliquots were added to a reaction mixture containing 1 mM MgSO4, 0.2 mM of each deoxynucleotide triphosphate, 1 U of Avian Myeloblastosis Virus reverse transcriptase, 1 U of thermostable DNA polymerase from Thermus flavus (Access RT-PCR System; Promega, Madison, WI), and 0.5 µM of the specific primers for the human IL-6 gene: 5'-TTCGGTCCAGTTGCCTTCT-3' (sense) and 5'-GTACTCATCTGGACAGCTC-3' (antisense).

These primers were localized in exon 2 (sense) and exon 4 (antisense), spanning two introns, so that signals from mRNA could be distinguished from those from possible contaminating DNA (40). Using these primers, PCR amplification yields a 416-bp product. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was coamplified, using specific primers for the human gene (41), as a constitutive control.

Total RNA and the primers were preincubated for 5 min at 65°C. Then, the reaction mixture (10 µl) was incubated for 45 min at 48°C, and 2 min at 95°C, followed by 30 to 32 cycles of 1 min at 95°C, 1 min at 60°C, and 2 min at 68°C, with a final extension of 7 min at 68°C. Controls without RNA and with known RNA were usually included in the PCR reaction. The PCR products were separated on 2% agarose gels, and bands were visualized by ethidium bromide staining and quantified by densitometric scanning (ImageQuant; Molecular Dynamics, Sunnyvale, CA). IL-6 PCR product densitometric values were normalized against those of the corresponding GAPDH product. To confirm the identity of the PCR products, they were purified by agarose gel electrophoresis and extracted with Wizard PCR prep columns (Promega). The extracted PCR products were then bidirectionally sequenced with a dye-terminator cycle-sequencing kit (Applied Biosystems, Branchburg, NJ), according to the manufacturer's instructions. Sequences were resolved on an ABI PRISM 310 automatic sequencer.

IL-6 Assay
hOB cells were incubated for 24 h in culture medium, in the presence or absence of the test agents. The conditioned medium was collected and kept at -20°C before assay. This medium was centrifuged to eliminate cell debris before IL-6 determination. IL-6 was measured by a sandwich-type enzyme-amplified sensitivity immunoassay (EASIATM, Medgenix; Fleurus, Belgium), using a blend of capture monoclonal antibodies against different IL-6 epitopes, and a horseradish peroxidase-labeled monoclonal anti-IL-6 antibody. The assay detects free IL-6, and has a sensitivity of 2 pg/ml. Cell protein was assayed in 0.1N NaOH-solubilized cell extracts, using the Bradford method (42). The data were expressed as the amount of IL-6 produced per mg cell protein.

Statistical Analyses
Results throughout the text are expressed as mean ± SD. The significance of the differences between groups was assessed by unpaired t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To examine the changes of IL-6 expression triggered by the agonists, we used semiquantitative RT-PCR. This technique detects relatively large changes of mRNA using conditions that provide submaximal amplification. Preliminary titration experiments showed that 0.1 to 1 ng of total RNA from either PTHrP-stimulated or nonstimulated (Figure 1) hOB cells, and 32 cycles, fit this requirement in our RT-PCR system. Using this maneuver, we found that 10 nM PTHrP (107-139) stimulated IL-6 mRNA, which peaked at 1 to 2 h, representing twofold over basal values, and returned to baseline thereafter, in hOB cells (Figure 2, A and C). PTHrP (1-34), at 10 nM, similarly increased IL-6 gene expression within the same time frame in these cells (Figure 2, B and C). A 10-fold higher concentration of these PTHrP peptides did not induce a higher stimulation of IL-6 mRNA in these cells (not shown).



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  		Figure 1. Titration curves for reverse transcription (RT)-PCR amplification of interleukin-6 (IL-6) (A) and GAPDH (B) mRNA, with specific primers and experimental conditions as mentioned in the text, using 32 cycles. Points are means obtained with total RNA isolated from nonstimulated osteoblastic cells from human trabecular bone of two different patients.

 


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  		Figure 2. Time-dependent IL-6 expression in osteoblastic cells from human trabecular bone. The cells were stimulated in serum-free medium for 0.5 to 6 h with either PTHrP (107-139) (A) or PTHrP (1-34) (B), at 10 nM, before RNA isolation and RT-PCR analysis. These results are representative of four different patients. (C) Changes in IL-6 mRNA levels, corrected to those of GAPDH mRNA, in these cells after stimulation with either PTHrP (107-139) or PTHrP (1-34), at 10 nM, for 2 h. Data are mean ± SD corresponding to 11 different patients. *P < 0.01, compared with nonstimulated control (100%).

 

The effect of the N-terminal region of PTH and PTHrP on IL-6 mRNA in osteoblastic cells has previously been shown to display features common to the early response gene family, including sensitivity to transcription and protein synthesis inhibitors (32,34). Hence, we next examined whether these inhibitors also affected the PTHrP (107-139)-induced IL-6 gene expression in hOB cells. We found that 10 µg/ml actinomycin D abolished the maximal effect of PTHrP (107-139), and that of PTHrP (1-34), at 10 nM, in these cells (Figure 3A). In contrast, 100 µg/ml puromycin superinduced IL-6 gene expression, mainly in the presence of either PTHrP peptide for a time period when their stimulatory effect was decreasing in these cells (Figure 3B).



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  		Figure 3. Effect of different agents on IL-6 expression induced by PTHrP (107-139) and PTHrP (1-34) in osteoblastic cells from human trabecular bone. The cells were treated in serum-free medium with each PTHrP peptide, at 10 nM, with and without either actinomycin D (ActD) (10 µg/ml) (A), for 2 h, or puromycin (Puro) (100 µg/ml), for 4 h (B). Act D and Puro were added immediately before the addition of PTHrP peptides. These results are representative of four different patients.

 

We next tested whether these effects of both PTHrP domains on IL-6 mRNA are reflected in IL-6 secretion by hOB cells. Figure 4 shows that PTHrP (107-139) or PTHrP (1-34) similarly and dose dependently stimulated twofold IL-6 into these cells' conditioned medium at 24 h. PMA, at 100 nM, was found to maximally increase fivefold IL-6 in the hOB cell-conditioned medium at the latter time period (Table 1). However, treatment of hOB cells with these PTHrP peptides, added together, at concentrations in the range of 10 nM to 1 pM, failed to induce a significantly higher stimulatory effect on secreted IL-6 compared to that observed with each peptide alone. Therefore, secreted IL-6 was 170 ± 18%, 163 ± 20%, or 162 ± 8% in hOB cells treated for 24 h with PTHrP (1-34) and PTHrP (107-139), PTHrP (107-139), or PTHrP (1-34) at 1 pM, respectively, compared to that in either untreated hOB cells (100 ± 16% or 20.7 ± 3.3 ng/mg protein) (P < 0.01) or in these cells treated with 10 nM PTHrP (38-64), a peptide devoid of known biologic activity in these and other cells (2,13), which was 104 ± 9% (n = 3).



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  		Figure 4. Effect of PTHrP (107-139) and PTHrP (1-34) on IL-6 secretion by osteoblastic cells from human trabecular bone. Serum-depleted cells were treated with different concentrations of each PTHrP peptide for 24 h. IL-6 in the cell-conditioned medium was determined by enzymeamplified sensitivity immunoassay (EASIATM) as described in Materials and Methods. Results are mean ± SD of values from eight different patients, performed in duplicate. These cells from five of these patients were tested for IL-6 mRNA, and found to respond to both PTHrP peptides. *P < 0.01, compared with nonstimulated control (100 ± 10%, or 15.9 ± 1.6 ng/mg protein).

 

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  		Table 1. Effect of different agents on PTHrP (107-139)-stimulated secretion of IL-6 by osteoblastic cells from human trabecular bonea
 

Our previous results indicate that the effects of PTHrP (107-139) on osteoblastic proliferation and various osteoblastic markers appear to be mediated by a PKC-dependent mechanism (12,13). We found that 100 nM BIM, a PKC inhibitor (43), in contrast to 100 nM H89, a PKA inhibitor (44), eliminated the IL-6 mRNA induction by 10 nM PTHrP (107-139) for 2 h in hOB cells (Figure 5). Moreover, 25 nM BIM, but not 25 µM RpcAMPS, another PKA inhibitor (45), also significantly decreased PTHrP (107-139)-stimulated IL-6 secretion by these cells at 24 h (Table 1).



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  		Figure 5. Effect of different agents on IL-6 expression induced by PTHrP (107-139) and PTHrP (1-34) in osteoblastic cells from human trabecular bone. The cells were treated in serum-free medium with each PTHrP peptide, at 10 nM, with and without different agents, for 2 h. Bisindolylmaleimide I (BIM; 100 nM) and N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89; 100 nM) were added 1 h before stimulation with each PTHrP peptide. These results are representative of two different patients.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The N-terminal region of both PTH and PTHrP stimulates bone resorption by inducing the production of osteoclast activators by osteoblasts (5). In this regard, although a previous study failed to show any effect of PTH (1-34) on IL-6 production in human osteoblastic cells (46), recent reports support a role of IL-6 as a mediator of the PTH-like osteoclastic effects of PTHrP (31-38).

In the present report, we found that PTHrP (1-34) induced a rapid and transient increase of IL-6 mRNA in human osteoblastic cells. This induction has features of an early gene response, consistent with previous findings in other osteoblastic cells in vitro, and mouse osteoblasts in vivo (32,34,35). We found that PTHrP (107-139) also stimulated IL-6 gene expression, with a response pattern similar to that observed with PTHrP (1-34), in these cells. Moreover, the increase of IL-6 mRNA triggered by both N- and C-terminal PTHrP domains was accompanied by an increase of immunoreactive IL-6 in the medium conditioned by hOB cells, which is in the same range as that observed for IL-6 mRNA. It is unlikely that this inducing effect of PTHrP (107-139) on IL-6 in osteoblastic cells is nonspecific since PTHrP (38-64), which lacks biologic activity (2,13), did not affect secreted IL-6 in hOB cells' conditioned medium.

A previous study found an inhibitory effect of PTHrP (107-111) and PTHrP (107-139) on rat osteoclasts in vitro, which was lower in the presence of osteoblasts (19). However, other investigators failed to find any consistent effect of the latter PTHrP peptides on bone resorption by isolated osteoclasts from rat, chicken, or mouse (20,21). In contrast, a recent report has shown a stimulatory effect of PTHrP (107-139) on osteoclastogenesis in isolated mouse bone cells (containing osteoblasts) and mouse spleen cells (in the absence of osteoblasts) (22). Thus, current data on the in vitro effects of C-terminal PTHrP in osteoclasts from various species are still inconsistent. Recently, an inhibitory effect of both bone resorption and bone formation indexes after injection of PTHrP (107-139) in the mouse calvaria for several days was reported (23). Interestingly, another histomorphometric study in osteopenic rats treated daily for 13 d with PTHrP (107-111) found an increased bone mass only at the cortical level. Meanwhile, the effect of this peptide on the trabecular bone of these animals was to decrease bone formation and increase bone resorption (24). The decrease in bone formation in these studies is consistent with the antiproliferative effect of this C-terminal PTHrP region on osteoblastic cells in vitro (12,13). Our finding herein that PTHrP (107-139) induces IL-6 in osteoblastic cells from trabecular bone is also consistent with one of the aforementioned studies (24), since IL-6 appears to stimulate osteoclast formation and activity, and inhibit bone formation both in vitro and in vivo (31,33,38,47). However, a definite cause—effect relationship between increased IL-6 and decreased osteoblastic growth and/or activity cannot be raised from this and other recent studies (48).

Our findings might be particularly relevant in chronic renal failure, where increased circulating C-terminal PTHrP fragments and IL-6 have recently been reported (49,50). In this regard, a recent study using in situ hybridization has shown that osteoclasts and bone marrow cells of patients on chronic maintenance dialysis express mRNA of IL-6 and its receptor (51). Interestingly, the latter mRNA in osteoclasts were increased, associated with high circulating PTH levels and the increased bone resorption (51). In the latter report, however, a weak signal for IL-6 or its receptor transcripts was detected in osteoblasts (51). Additional studies are necessary to clarify the role of IL-6, and its interaction with systemic PTH and various PTHrP fragments in the bone microenvironment, on renal osteodystrophy.

The promoter region of the IL-6 gene contains cAMP response elements, and an NF-{kappa}B-binding site that confers PKC inducibility to this gene (52,53). The stimulatory effect of the N-terminal region of PTH and PTHrP on IL-6 production in osteoblastic cells appears to depend at least in part on cAMP signaling (33,34,35,37). On the other hand, IL-1-induced IL-6 production occurs through PKC activation in human bone marrow stromal cells, where PMA also stimulates IL-6 (54). In the present study, we found that a PKC inhibitor, but not two PKA inhibitors, significantly decreased the stimulatory effect of PTHrP (107-139) on IL-6 mRNA and protein in hOB cells. Moreover, PMA, a PKC stimulator (55), increased IL-6 secretion in these cells. In this regard, both PTHrP (107-111) and PTHrP (107-139) stimulate PKC in human keratinocytes, rat spleen lymphocytes, and rat osteosarcoma cells ROS 17/2.8, within a dose range of pM to nM (55,56,57). Moreover, we have recently demonstrated that PTHrP (107-139) was unable to increase PKA activity in osteoblast-like osteosarcoma cells UMR 106 and hOB cells (12,13). In contrast, calphostin C, a PKC inhibitor, abolished the inhibitory effect of the latter agonist on these cells' growth (12,13). Furthermore, PKC activation seems to be involved in the aforementioned osteoclastic effects of PTHrP (107-139) (19,22). Taken together, these findings and the results herein support that PKC activation is involved in the PTHrP (107-139)-induced IL-6 gene expression in human osteoblastic cells.

We found that both PTHrP (107-139) and PTHrP (1-34), added together at various concentrations, induced the same stimulatory effect on IL-6 secretion as that of each peptide alone in hOB cells. Since PKA and/or PKC appear to be involved in IL-6 stimulation by these PTHrP peptides, the latter finding could be explained by cross-talk in both responsive signal transduction pathways in these cells, as suggested in other studies (14,58).

In summary, the present study in human osteoblastic cells supports a role for the C-terminal PTHrP region, similar to that of its N-terminal domain, as a putative stimulator of bone resorption through its effect on osteoblastic IL-6 expression.


   Acknowledgments
 
Acknowledgments

We thank Dr. L. Munuera from the Department of Orthopedics and Accident Surgery, Hospital La Paz, for providing us with the bone samples. We also thank A. Cebrián, from the Department of Genetics, Fundación Jiménez Díaz, for sequencing the IL-6 PCR product. Drs. Martínez-Fernández, Rodrigo, and Valín are fellows of Fundación FAES, Fondo de Investigación Sanitaria of Spain, and Fundación Conchita Rábago, respectively. This work was supported in part by grants from Fondo de Investigación Sanitaria of Spain (FIS 96/1167 and 97/0307) and Comisión Interministerial de Ciencia y Tecnología (CICYT, MAT 95/0249).


   Footnotes
 
American Society of Nephrology


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication February 26, 1998. Accepted for publication October 14, 1998.




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