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Endocrinology Vol. 138, No. 9 3918-3924
Copyright © 1997 by The Endocrine Society

ARTICLES

Parathyroid Hormone Increases Circulating Levels of Fibronectin in Vivo: Modulating Effect of Ovariectomy1

Ben hua Sun, Maryann Mitnick, Charlotte Eielson, Gang Quing Yao, Indu Paliwal and Karl Insogna

Departments of Internal Medicine, Surgery, and Comparative Medicine, Yale School of Medicine,New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Ben hua Sun, M.D., Endocrine Section, Fitkin I, P. O. Box 208020, Yale School of Medicine, New Haven, Connecticut 06520-8020.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To explore the effect of PTH on circulating levels of fibronectin (FN), adult female Sprague-Dawley rats were implanted with Alzet minipumps prepared to deliver 7 pmol/h·kg BW of either human PTH (1–34) or human PTH (1–84). Both forms of the hormone led to significant and progressive increases in circulating levels of FN over the 72-h study period (P < 0.001). However, at every time point, circulating levels of FN with human PTH (hPTH) (1–84) infusion were significantly higher than with hPTH (1–34), such that at the end of the infusion, mean levels in the hPTH (1–34) group were 32.2 ± 1.4 ng/ml, in the hPTH (1–84) group 93.8 ± 5.4 ng/ml, and in the vehicle infused group 14.6 ± 0.7 ng/ml. The greater agonist efficacy of hPTH (1–84) was not explained by differences in circulating levels of the hormones, and both forms of the hormone were equipotent at stimulating cAMP production by ROS 17/2.8 cells. However, hPTH (1–84) remained a more effective agonist than hPTH (1–34) at stimulating FN production in these cells (P < 0.001).

Nephrectomy did not blunt the ability of PTH to increase circulating FN in vivo, indicating that the kidney was not the source of the FN produced in response to PTH. Pretreament with the potent bisphosphonate APD to block bone resorption also did not blunt the in vivo response to PTH. Parathyroidectomy did not blunt the response. Cultured fetal rat bones showed a significant 2.4-fold increase in FN production when treated with PTH.

Consistent with our earlier in vitro studies , estrogen deficiency, induced by ovariectomy, significantly diminished the ability of PTH to increase circulating FN levels in vivo (P < 0.001).

We conclude that PTH increases circulating levels of FN in vivo and may be a physiologic regulator for the plasma form of this glycoprotein. The effects of PTH on circulating FN may reflect the anabolic properties of the hormone in bone and the blunted response following estrogen withdrawal could be a manifestation of the diminished bone formation vis-à-vis resorption seen in the estrogen deficient state.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
EXTRACELLULAR matrix proteins serve, in part, to direct the processes of cell growth and differentiation. Fibronectin (FN) is a large extracellular matrix glycoprotein with a structure and function determined by alternative splicing of a primary gene transcript, as well as by posttranslational modifications (1, 2). It is known to be involved in cellular processes such as cell adhesion, differentiation, and migration (1, 2, 3, 4, 5, 6).

The precise role FN plays in bone is unclear. The protein, however, is present throughout organogenesis and may represent an early scaffolding for collagen, as well as participate in recruitment and differentiation of bone cell precursors (3, 4, 5, 7, 8). Osteoblasts, chondrocytes, and mesenchymal cells produce FN (3, 9). Because FN is present throughout developing bone, this protein may participate in the recruitment of osteoblast progenitor cells (3, 4, 5). Studies have demonstrated osteoinductive factors in bone matrix between 100,000 and 300,000 daltons in size, and FN is believed to be one such factor (10). FN also contains collagen binding sequences, enabling it to act as an organizing framework for collagen (8, 11). In this context, it is noteworthy that immunohistochemical studies in bone demonstrate the presence of FN in differentiating rat osteoid (3, 7).

FN contains the amino-acid sequence ARG-GLY-ASP (RGD), which can interact with the integrin receptors expressed on osteoblasts (12) and osteoclasts (13). Osteoblasts appear to attach to FN through adherence to both the RGD tripeptide and heparin binding domains of FN (10). Further, mouse calvarial cell attachment to type I collagen is enhanced by FN (14). Osteoclast attachment through RGD sequences appears to be critically important for bone resorption because inhibition of RGD-directed osteoclast binding leads to inhibition of resorption by these cells (15, 16, 17). Interestingly, adhesion of osteoclast-like cells to both osteocalcin and laminin induces the production of FN by these cells (18, 19). It has been speculated that this matrix-induced FN production may provide an attachment site for osteoclasts at some stage in their differentiation process (18). Collectively, these data suggest that FN plays a role both in modeling of developing bone and in remodeling of mature bone.

Alternative splicing gives rise to cellular and plasma forms of FN (2, 20, 21). A variety of growth factors and cytokines have been reported to regulate cellular FN, and, in skeletal tissue, we have recently reported that PTH stimulates FN production by cultured primary human and rat bone cells and by a human osteosarcoma cell line (22). Further, withdrawal of 17ß-estradiol specifically diminished PTH-induced FN production by these cells. In contrast to these data, virtually nothing is known about factors that regulate plasma FN. In part, circulating FN appears to reflect general nutritional status (23). Most of the plasma form of FN is thought to be produced by the liver, (24, 25); however, an isolated rabbit lung preparation exposed to an oxidative challenge with H2O2 has been to shown to release FN suggesting the possibility that lung may also be source of circulating FN in vivo (26). No reports have addressed bone as a potential source of circulating FN.

In the present study, we extend our in vitro observation that estrogen modulates PTH-induced FN production. We report that PTH treatment dose dependently increases circulating levels of FN in vivo in rats and that this effect is not diminished by nephrectomy or by inhibiting bone resorption. However, in a manner analogous to the findings in vitro, ovariectomy significantly diminishes PTH-induced increases in circulating FN in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human PTH (hPTH) (1–84) and hPTH (1–34) were purchased from Bachem Inc. (Torrance, CA). Ham’s F12 cell culture medium, penicillin/streptomycin, isobutyl methylxanthine, L-cysteine-HCl and FBS were purchased from Sigma Co. (St. Louis, MO). Alzet minipumps were purchased from Alzet Inc. (Palo Alto, CA). Nembutal (50 mg/ml) was purchased from Abbott Laboratories (North Chicago, IL). Metophane was purchased from Pittman Moore Inc. (Mundelein, IL). APD (disodium (3-amino-1-hydroxypropylidene) bisphosphonate was a generous gift from Ciba-Geigy Corp. (Summit, NJ). Plasticware for tissue culture was purchased from Becton Dickenson Labware (Lincoln Park, NJ).

Preparation of minipumps
Alzet minipumps were loaded with hPTH (1–84) or (1–34) to deliver a dose of 7 pmol PTH/h·kg BW. The peptides were dissolved in 2% L-cysteine-HCl at a pH of 1.5, loaded into the pumps and the loaded pumps equilibrated at 37 C overnight before implantation.

Animals and surgical procedures
Female Sprague-Dawley rats (250–300 g) unmanipulated, ovariectomized, sham ovariectomized, and parathyroidectomized were purchased from Taconic Inc. (Germantown, NY). All animals were fed standard rat chow with free access to water. Ovariectomized and parathyroidectomized rats were studied one week after surgery.

Bilateral nephrectomy was accomplished via a midline abdominal incision after anesthetizing the animals with intraperitoneal Nembutal (50 mg/kg bw). The renal pedicles were identified and ligated using monofilament sutures and the kidneys removed. During the procedure each animal received approximately 6 ml of 0.9% sodium chloride through the left internal jugular vein to compensate for blood loss during the procedure. The abdominal wall was closed with monofilament sutures and the skin closed with surgical staples. The animals were allowed to recover from anesthesia and studied over the next 24 h.

APD pretreatment was accomplished by sc administration of 0.1 mg APD/kg bw in saline, 24 h before minipump implantation.

Minipumps were implanted sc in the suprascapular region after satisfactory anesthesia was accomplished with Metophane. The skin was closed with monofilament sutures.

Blood samples were obtained via the tail vein. Serum was separated and frozen at -70 C until assayed. These studies were approved by the Yale Animal Care and Use Committee.

Cell culture
ROS 17/2.8 cells were grown and passaged as previously reported (27). For determination of the effect of PTH on FN production, cells were grown in 6-well plates and studied at 14 days post confluence. Cells were treated for 72 h with either hPTH (1–34), hPTH (1–84) or vehicle in Ham’s F-12 culture medium containing 1% FCS, the media harvested, clarified by centrifugation and stored at -70 C until assayed.

Cellular cAMP content was determined in cells grown in 24-well plates and studied at 14 days post confluence. Cell were pretreated for 10 min at 37 C with IBMX and then with PTH for 10 min as previously reported (28) with the following modifications. The cells were not prelabeled with 3[H] adenine and the reaction was stopped by adding 0.4 ml of ice cold 90% n-propanol to each well. Cells were extracted with n-propanol overnight at -70 C and the extracts assayed the following day for cAMP as detailed below. Cell number was determined using a hemocytometer.

Cultured fetal rat long bones
Long bones, isolated from day 19 rat fetuses, were isolated and cultured as previously reported (29). Each bone was cultured in a final volume of 400 µl, and the FN content of the media determined after 72 h of treatment with either vehicle or PTH.

Determination of cellular cAMP
Cellular extracts were dried under nitrogen and reconstituted with 250 µl of sodium acetate, 0.5 M, pH 6.2 (assay buffer). A 100 µl aliquot of the reconstituted sample was diluted 1:5 with assay buffer and 100 µl taken for acetylation. Acetylation was accomplished by adding 5 µl of 2:1 triethylamine:acetic acid for 3 min at room temperature (30). The reaction was stopped by adding 900 µl of assay buffer. Modified assay buffer was prepared by adding 50 µl of the acetylation reagent (2:1 triethylamine:acetic acid) to 10 ml of assay buffer. Modified assay buffer is used for the preparation of the standards and in the blank and zero standard tubes.

Acetylated samples were then assayed using a double-antibody RIA that uses a prereacted antibody complex as previously published (31). The intraassay coefficient of variation is 2.6%. The interassay coefficient of variation is 3.7%.

Determination of serum calcium and 1,25(OH)2vitamin D
Serum calcium was determined by atomic absorption spectrophotometer. 1,25(OH)2vitamin D levels were determined as previously reported (32).

Assays for human PTH (1–84) and (1–34)
Serum levels of hPTH (1–84) were determined using the Allegro intact PTH(1–84) two-site immunoradiometric assay (Nichols Institute, San Juan Capistrano, CA). The sensitivity of the assay is 1 pg/ml. Intra and interassay coefficients of variation are 3.4% and 4.6%, respectively.

Serum levels of hPTH (1–34) were determined using a nonequilibrium immunoassay that employs a goat antiserum to hPTH (1–34) (kindly provided by Lawrence Mallette, M.D., Ph.D., VA Medical Center, Houston, TX), 125I-hPTH (1–34) as radioligand and dextran-coated charcoal for phase separation. Human PTH(1–34) is iodinated using the chloramine-T method (33) and purified by reverse phase HPLC on a Vydac C18 column. The standard curve is run in human hypoparathyroid serum or charcoal-stripped human plasma to control for serum effects. Nonspecific binding tubes are run for each unknown. Standards or unknown samples in 100 µl of Veronal buffer (0.05 M sodium barbital pH 8.6) are incubated with 100 µl of antisera (1:6,000 dilution in Veronal buffer) for 48 h at 4 C. 4,000 cpm of 125I-hPTH (1–34) is then added in 100 µl of Veronal buffer and the incubation continued for 48 h. Two hundred microliters of freshly prepared, dextran-coated charcoal is then added and the incubation continued on ice for 45 min. The samples are then spun at 4,800 x g at 4 C and both the supernatant and pellet counted. The standard curve is plotted as % B/Bovs. pg/ml of hPTH (1–34). The interassay coefficient of variation is 4.1%, and the intraassay coefficient of variation is 3.2%.

FN assay
The concentrations of FN in serum and conditioned media were determined using a commercially available FN immunoassay kit with a rat FN standard (Biomedical Technologies Inc, Stoughton, MA) as previously reported (22). The primary antibody is rabbit antihuman FN used at a final titer of 1:40,000. Goat antirabbit IgG is employed as the second antibody.

Statistical analyses
Two-way or one-way ANOVA was used for all analyses except for the analysis of FN production by cultured fetal rat bones where an unpaired t test was used. Statistical analyses were performed using the SuperANOVA software package version 1.11 (Abacus Concepts Inc. Berkeley, CA) for Macintosh. All data are presented as mean ± SE of the mean.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
All animals tolerated the procedures well. There were no deaths during any of the studies.

Effect of hPTH (1–34) and hPTH (1–84) infusion on calcium levels
As shown in Table 1Go, infusion of either hPTH (1–34) or hPTH (1–84) for 72 h at a constant rate of 7 pmol/h did not influence serum calcium levels. The mean serum calcium value in the vehicle infused group at the end of the study period was 9.9 ± 0.1 mg/dl, vs. 10.0 ± 0.1 mg/dl in both the hPTH (1–34) and hPTH (1–84) groups.


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  		Table 1. Effect of PTH infusion for 72 h on serum levels of calcium, PTH and 1,25(OH)2D

 
Effect of PTH infusion on circulating levels of FN
Infusion with hPTH (1–84) or hPTH (1–34) led to significant increases in circulating concentrations of FN (Fig 1Go). At 72 h, FN levels in the hPTH (1–84) group were elevated 6.4-fold and in the hPTH (1–34) group 2.2-fold over values in the vehicle infused group (93.8 ± 5.4 and 32.2 ± 1.4 vs. 14.6 ± 0.7 ng/ml, respectively). For the hPTH (1–84) group, this effect was significant at 24 h of infusion at a time when circulating levels of hormone were well within a physiologic range (5.9 ± 0.4 pM). As is also apparent from the data, hPTH (1–84) has significantly greater agonist efficacy than the shorter form of the hormone (P < 0.001). At every concentration examined, mean levels of serum FN were highest in the hPTH (1–84) group. Vehicle infused animals showed no change in FN levels over the 72-h time course.



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  		Figure 1. Effect of hPTH (1–34) and hPTH (1–84) on circulating FN levels in rats. The dose-response curve for (1–84)hPTH is significantly different from that for (1–34)hPTH [P < 0.001; n = 11 for the (1–34)hPTH group, n = 11 for the (1–84)hPTH group and n = 11 for the vehicle-infused group].

 
To determine if the greater efficacy of hPTH (1–84) was due to higher circulating concentrations of this form of the hormone, serum levels of (1–84) and hPTH (1–34) were measured during the 72-h infusions. Throughout the infusion, levels of hPTH (1–34) were higher than those for hPTH (1–84). Thus, as shown in Table 1Go, mean levels of hPTH (1–34) were two times higher than those of hPTH (1–84) at the end of the 72-h study period (24.2 ± 1.6 vs. 11.6 ± 0.8 pg/ml, P < 0.001 for effect of peptide length). Therefore, despite higher circulating levels, hPTH (1–34) was less effective an agonist. Because differences in circulating levels of the hormone did not explain the greater efficacy of hPTH (1–84) on FN in vivo, we next explored the possibility that this reflected differences in intracellular signaling pathways.

Effect of hPTH (1–34) and hPTH (1–84) on FN and cAMP production by cultured bone cells in vitro
The principal signaling pathway for PTH is via activation of adenylate cyclase and protein kinase A. We therefore examined the effect of the two forms of the hormone on cAMP generation in a well characterized rat osteosarcoma cell line (ROS 17/2.8). Over the concentration range 5 x 10-11 to 1 x 10-7 M, hPTH (1–84) and hPTH (1–34) were equipotent at stimulating cAMP production by these cells (Fig. 2Go, left panel; P = NS for effect of peptide length). Thus at a concentration of 1 x 10-9, treatment with both hormones led to equivalent amounts of cAMP accumulation [196 ± 1 vs. 205 ± 3 pg/ml; hPTH (1–84) vs. (1–34)]. Despite this, hPTH (1–84) was a more effective stimulant of FN production in vitro than hPTH (1–34) over the entire concentration range examined (Fig. 2Go, right panel; P < 0.001 for effect of peptide length). At a concentration of 1 x 10-9 M, hPTH (1–84) stimulated 1.5-fold more FN production than hPTH (1–34) (382.5 ± 13.5 vs. 247.5 ± 3.6 ng/ml, respectively). These in vitro findings mirror the in vivo results and suggest intrinsic differences in the ability of these two forms of the hormone to simulate FN production. Because both forms of the hormone bind with equivalent affinity to the PTH receptor (34), and given their equal ability to activate adenylate-cyclase, the differences in effects on FN production must result from other differences, as discussed below.



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  		Figure 2. Effect of (1–34)hPTH and (1–84)hPTH on cAMP generation (left panel) and FN production (right panel) in ROS 17/2.8 cells. Each data point is the mean of four determinations. Each determination was measured in duplicate. The graphs depict the data from three independent experiments. The two dose-response curves for cAMP accumulation are not different from each other (P= NS). The two dose-response curves for FN production are significantly different (P < 0.001). Neither treatment affected cell number (P= NS). The average number of cells per well was 2.1 x 106.

 
PTH increases circulating levels of 1,25 dihydroxyvitamin vitamin D [1,25(OH)2D]
As seen in Table 1Go, 1,25(OH)2 vitamin D levels were also elevated to a greater extent in the hPTH (1–84) group. After 72 h of infusion mean, levels of 1,25(OH)2D were 81 ± 2 pg/ml in the hPTH (1–84) group, 65 ± 1 pg/ml in the hPTH (1–34) group and 48 ± 1 pg/ml in the vehicle infused group (P < 0.001).

Effect of PTH infusion on circulating FN levels in parathyroidectomized rats
In parathyroidectomized rats, 72 h of hPTH (1–84) infusion increased circulating levels of FN from a mean value of 25.2 ± 1.0 ng/ml at baseline to a value of 96 ± 13.4 ng/ml at the end of the infusion (n = 4). The value at 72 h is comparable to the mean circulating FN level observed in hPTH (1–84) infused intact rats (93.8 ± 5.4 ng/ml). In vehicle-infused parathyroidectomized animals, circulating levels of FN did not change over the 72 h time course 25.2 ± 1.0 ng/ml -> 26.8 ± 1.0 ng/ml (n = 4).

Estrogen modulates PTH-induced increases in circulating FN
Ovariectomy diminished the ability of hPTH (1–84) to increase serum FN. As shown in Fig. 3Go, there was no difference in basal levels of FN between sham and ovariectomized animals but at every time point over the 72-h infusion period, the effect of PTH was significantly less in the estrogen deficient animals (P < 0.001). At 72 h there was a 1.6-fold reduction in mean serum FN levels in ovariectomized animals as compared with sham-operated controls (39.5 ± 2.0 vs. 64.7 ± 2.7 ng/ml). There was no effect of ovariectomy on circulating levels of PTH (P = NS for treatment group effect). These in vivo findings precisely recapitulate our previously reported findings in cultured bone cells where estrogen withdrawal was found to attenuate the stimulatory effect of PTH on FN production (22).



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  		Figure 3. Effect of ovariectomy on the ability of hPTH (1–84) to increase circulating FN levels in vivo. Sham operated and OVX animals were operated upon one week before study. All animals were infused with hPTH (1–84) for 72 h and FN levels measured in the serum every 24 h during the course of the infusions. The two curves are significantly different (P < 0.001; n = 6 for the OVX group and n = 6 for the sham group).

 
Effect of nephrectomy and in vivo inhibition of bone resorption on PTH-induced changes in circulating FN
The principal target tissues for PTH are kidney and bone. The observed increase in circulating FN in response to PTH could reflect protein originating entirely or in part from the kidney. However, nephrectomy did not blunt the ability of hPTH (1–84) to increase serum FN levels. After 24 h of PTH treatment, unmanipulated animals demonstrated a 300 ± 6% increase in FN levels over baseline, whereas nephrectomized animals demonstrated a 537 ± 13% increment (Fig. 4Go). The 537 ± 13% increment in circulating FN levels seen after 24 h of PTH infusion reflects an increase over the values obtained in nephrectomized rats infused with vehicle for 24 h. Therefore, a decline in FN clearance due to nephrectomy is not the explanation for the findings.



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  		Figure 4. Percent increase in circulating FN levels following 24 h of treatment with hPTH (1–84) in unmanipulated animals (left), nephrectomized animals (center) and APD-pretreated animals (right). Percent increase was calculated relative to the FN level following 24 h of vehicle infusion in the control groups. Vehicle-infused, unmanipulated animals served as controls for the unmanipulated animals treated with hPTH (1–84) (n = 11 for the hPTH (1–84) infused group, n = 11 for the vehicle infused group). Mean serum levels of FN at 24 h were 15.1 ± 2.5 vs. 45.3 ± 8.4 ng/ml, vehicle-infused vs. PTH-infused. APD-pretreated, vehicle-infused animals served as controls for the APD group, (n = 5 for the APD-pretreated, PTH-infused group and n = 4 for the APD-pretreated, vehicle-infused group). Mean serum levels of FN at 24 h were 25.5 ± 5.8 vs. 82.6 ± 9.8 ng/ml, vehicle-infused vs. PTH-infused. Vehicle-infused, NPX animals served as controls for the NPX group, (n = 3 for the NPX, PTH-infused group and n = 2 for the NPX, vehicle-infused group). Mean serum levels of FN at 24 h were 18.7 ± 2.5 vs. 100.5 ± 26.3 ng/ml, vehicle-infused vs. PTH-infused.

 
Because PTH stimulates resorption as well as formation in bone, we next sought to determine if PTH-mediated bone resorption was responsible for the observed increases in FN. That is, there are two ways by which PTH, acting in bone, could increase FN production in that tissue. PTH could directly stimulate osteoblasts to produce FN as we have shown in vitro, or PTH may induce bone resorption and the observed increase in FN could, in this case, reflect release from degraded bone matrix. We therefore pretreated animals with the potent bisphosphonate APD at a dose previously reported to block bone-resorption in vivo (35) and assessed the effect of this maneuver on hPTH (1–84)-induced FN production. APD-treated animals demonstrated a 324 ± 5% increase in FN levels following 24 h of PTH infusion, comparable with the 300 ± 16% increase observed in control animals treated with PTH (Fig. 4Go).

PTH stimulates FN production from fetal rat long bones
We next examined the effect of 10-8 M PTH on FN production from fetal rat long bones. After 72 h of culture, the concentration of FN in the media from the PTH treated bones was 2.3-fold higher than in the vehicle treated bones (650 ± 30 vs. 280 ± 10 ng/ml; n = 5 for both groups; P < 0.001; two-tailed t test).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The principal findings of the current study are that: 1) PTH increases circulating levels of FN in vivo. 2) Human (1–84)PTH is more effective than hPTH (1–34) at increasing circulating FN. This is not due to differences in receptor affinity or ability to stimulate protein kinase A. 3) Estrogen deprivation blunts the ability of hPTH (1–84) to increase FN levels in vivo, but neither nephrectomy nor inhibition of bone resorption affect the response. 4) PTH stimulates cultured fetal rat bones to produce FN.

The ability of any systemic hormone to increase circulating levels of FN has not, to our knowledge, been previously reported. The exact tissue source of the FN cannot be definitively established from these studies but several lines of evidence suggest that bone may be an important source. The principal sites of PTH action in the mature animal are in bone and kidney (36). Because nephrectomy does not blunt the ability of PTH to increase circulating FN levels, the kidney is unlikely to be the source of FN. However, PTH receptors are expressed in several tissues in adult rats including the liver (37), and it is possible that the observed effects of PTH are due to changes in hepatic FN synthesis.

Evidence for a skeletal FN source is provided by our previous report that PTH directly stimulates primary cultures of rat osteoblasts to produce FN (22). Further, in the current study, we observed that PTH stimulates FN production from bone organ cultures. In previous in vitro studies (22), we were unable to demonstrate a change in steady-state FN mRNA levels after PTH treatment, and therefore the precise mechanism for the agonist effect is unclear. It is possible that PTH, as has been reported for certain cytokines (38), changes the splicing pattern of FN transcripts and that PTH stimulates expression of the plasma FN splice variant. However, this is unlikely to be the sole mechanism for the observed effect because, in vitro, total FN production (i.e. both cell-associated and soluble) is increased by PTH (22). It is also possible that PTH changes the clearance of FN but the direct agonist effect of PTH both in cultured bone cells and in bone organ culture argues that a direct stimulatory effect on production is more likely. Because PTH has anabolic properties (39), the ability to stimulate synthesis of an important early matrix protein in bone may, in part, reflect this capability. It will be of interest to determine if FN is a useful in vivo marker when PTH is used as a therapeutic agent.

The difference in agonist efficacy for hPTH (1–84) and hPTH (1–34) is interesting. Differences in circulating levels of the two forms of the hormone do not support the notion that differential clearance is the basis for their differing efficacy because hPTH (1–34) levels were somewhat higher during the infusion studies. Previous work has documented that both forms of the hormone bind with equal affinity to the PTH receptor in bone (34). Our in vitro data indicate that the ability to generate cAMP was equivalent for both forms of the hormone (Fig. 2Go, left panel). Therefore, differential effects on alternate signaling pathways, such as protein kinase C-dependent pathways, may be responsible for this effect. In support of this notion is the work of van Leeuwen et al. who have shown that (1–84) PTH induced a 50% greater increase in cytosolic calcium than (1–34) PTH in osteoblast-like cells and that the increase was more sustained with the full length hormone (40). The recent characterization of a novel receptor, specific for the carboxyterminus of (1–84) PTH, also suggests that this region of the hormone may have unique effects not shared by truncated forms of the protein (41). For example, Murray et al. have reported that (53–84) hPTH stimulates alkaline phosphatase activity in dexamethasone treated osteoblast-like cells (42) and (52–84) hPTH stimulates collagen production by chondrocytes (43). These findings may also be relevant to the greater increase in circulating levels of 1,25 (OH)2 vitamin D, noted in response to hPTH (1–84) vis-à-vis hPTH (1–34) in the current study. Conversely, it is possible that although the two forms of the hormone appear equipotent for cAMP generation in vitro, they may have different in vivo efficacy in bone. Our data do not address this possibility.

The potent bisphosphonate APD did not effect the ability of PTH to increase circulating FN arguing against release of matrix FN via PTH-enhanced bone resorption. It is possible, however, that PTH induction of neutral proteases in osteoblasts could degrade matrix FN, despite inhibition of osteoclastic activity because some of these proteases have been reported to cause release of FN fragments from other connective tissues (44, 45). However, in this case the released FN is degraded, and it is unclear if our immunoassay would recognize these fragments.

The effect of ovariectomy parallels our in vitro findings that estrogen deprivation diminishes the ability of bone cells to produce FN in response to PTH. Thus, in ovariectomized rats, there was a significant diminution in the ability of PTH to increase circulating levels of FN. Estrogen is another hormone with clear trophic effects in bone. Estrogen has been reported to stimulate the in vitro production of several extracellular matrix proteins including collagen and TGF-ß (46). This effect may be predominantly in endosteal bone because estrogen has been reported to have an inhibitory action on collagen, osteopontin, and osteonectin production in periosteal osteoblasts (47, 48). A diminished ability of PTH to stimulate FN production in the absence of estrogen might be one mechanism for the relative uncoupling of bone-turnover seen in the setting of estrogen deprivation. Thus, reduced FN production might lead to diminished matrix deposition, diminished bone cell recruitment, and ultimately a relatively decreased rate of bone formation in the estrogen deprived state. In this context, it is of interest that osteoporotic women have recently been found to have lower serum levels of FN than aged matched controls (49).

In summary, these findings suggest a potentially important stimulatory role of PTH on circulating FN levels in vivo. This effect is modulated by estrogen such that in the absence of estrogen, the effect is diminished. This interaction may reflect in vivo changes in bone and, if so, could have important implications for the anabolic effects of both of these bone-active hormones. It will be of interest to examine the effects of intermittent PTH treatment on circulating levels of FN and to determine whether these findings extend to humans.


   Acknowledgments
 
We wish to thank Ms. Gunilla Thulin for expert technical assistance in performing the nephrectomies and Drs. Thomas Carpenter, Eleanor Weir and John Orloff for their helpful reviews of the manuscript.


   Footnotes
 
1 The work was supported by Grant AR-39571 from the National Institutes of Health (to K.L.I.) and by a grant from the Patrick and Catherine Weldon Donaghue Medical Research Foundation (to K.L.I.). Back

Received January 17, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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