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Endocrinology Vol. 139, No. 3 974-981
Copyright © 1998 by The Endocrine Society

ARTICLES

In Vivo Anabolic Effects of Parathyroid Hormone (PTH) 28–48 and N-Terminal Fragments of PTH and PTH-Related Protein on Neonatal Mouse Bones1

Souheir Rihani-Bisharat, Gila Maor and Dina Lewinson

Division of Morphological Sciences, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, 31096 Haifa, Israel

Address all correspondence and requests for reprints to: Dina Lewinson, Division of Morphological Sciences, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, P.O.B. 9649, 31096 Haifa, Israel.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We developed a neonatal mouse model to investigate in vivo anabolic effects of intact PTH (1–84) and its two fragments PTH (1–34) and PTH (28–48) and of the N-terminal fragment of PTH-related peptide [PTHrP (1–34)]. Two-day-old mice were injected with low-dose (0.05 µg/g body weight) and high-dose (0.2 µg/g body weight) of each of these peptides daily for 6 or 16 consecutive days. Long bones (tibias and femurs) and mandibular condylar cartilages were harvested. Total DNA and protein were analyzed as parameters for anabolic effects. DNA was increased significantly in tibias only by low doses of PTH (1–84) and PTH (1–34), but by both doses of PTH (28–48). In the cartilages of the mandibular condyles, both doses of all three peptides increased DNA. Total protein was increased in the tibia by the low dose of the three peptides, whereas in the condylar cartilage high doses of PTH (1–34) and PTH (28–48) also caused a 2- to 4-fold increase. When the effects of PTH (1–34) and PTHrP (1–34) on the tibias were compared, it became apparent that PTH (1–34) was more effective than PTHrP (1–34) when injected in low doses, but the latter caused a severalfold increase in DNA and protein at both doses. The outstanding anabolic effect of PTH (28–48) was further investigated using [3H]thymidine autoradiography, analysis of insulin-like growth factor I (IGF-I) protein, and localization of IGF-I messenger RNA (mRNA) by in situ hybridization. PTH (28–48) increased by 3-fold the number of [3H]thymidine-labeled cells in the epiphyseal cartilage of tibias removed from 8-day-old injected mice, and in the proliferative zone of the epiphyseal growth plate of tibias removed from 18-day-old injected mice. Femurs from the latter showed a 20% increase in their IGF-I content. In parallel, only tibias from 18-day-old injected mice showed IGF-I mRNA localization in proliferating chondrocytes, whereas those from vehicle-injected control mice did not exhibit IGF-I mRNA. In summary, our study showed that the neonatal mouse is a sensitive model to examine anabolic effects of different PTH and PTHrP fragments. It also revealed that PTH (28–48) has strong anabolic effects on this model, and suggests that IGF-I might mediate the anabolic effects of PTH (28–48).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANIMAL as well as clinical studies support the view that PTH is an anabolic factor for bone, especially for cancellous bone. When administered intermittently, it prevents bone loss and stimulates bone formation in aged immobilized or ovariectomized rats, and increases bone mass in postmenopausal women as well as in osteoporotic men (1, 2). Although most in vivo studies have looked for the anabolic effects of intact PTH or its fragments on aged or sexually retired animals or humans, fewer studies have explored their effects on very young and still-growing animals (1, 2, 3, 4, 5, 6).

Another peptide, the PTH-related protein (PTHrP), which was discovered as a result of the search for the circulating factor responsible for humoral hypercalcemia of malignancy, has recently been found to be expressed widely in fetal skeletal and extraskeletal tissues concomitantly with its receptor. After birth, PTHrP expression disappears from most neonatal tissues, its expression being limited to tissues such as pancreas, mammary gland during lactation, and maternal-fetal placental complex, but especially in malignant tissues (for review, see Ref.7). Whereas PTH functions as a systemic regulator of calcium homeostasis through its actions on receptors expressed in bone and kidney, PTHrP has been postulated to function mainly as a paracrine/autocrine regulator of cellular growth and differentiation through actions mediated by activation of the common receptor it shares with PTH. The striking homology of the 1–13 N-terminal portion of both PTH and PTHrP enables their common activation of the same receptor (8). Moreover, the targeted disruption of the PTHrP gene is lethal and has been found to be associated with skeletal dysplasia in homozygous mutants (9) and with abnormal cartilage development and altered endochondral bone formation (10, 11).

In endochondral bones, the two genes responsible for both PTHrP and PTH/PTHrP receptor are expressed distinctly but very closely spatially and temporally during development, consistent with the hypothesis that PTHrP acts as a paracrine/autocrine peptide factor. A common receptor mediates both the paracrine/autocrine PTHrP signal and the endocrine PTH signal. By the end of the fetal period in the rat, PTHrP messenger RNA (mRNA) can be found to be highly expressed in osteoblasts in the primary ossification center, along the bone collar of the tibia, and in maturing chondrocytes of the growth plate, concomitantly with the expression of PTH/PTHrP receptors, which can also be demonstrated in the 4-week-old rat (12, 13). PTHrP has likewise been demonstrated immunohistochemically in fetal and young tibial epiphyseal cartilage of the rat (14).

Although evidence for the role of PTHrP in the regulation of endochondral bone formation has been accumulating, there are little available data on the involvement of PTH in endochondral bone formation in the neonatal period (15, 16). Now that the PTH receptor has been localized to maturing chondrocytes of the growth plate (13), we hypothesize that PTH and/or PTHrP might be involved in the regulation of growth in the neonatal period. To our best knowledge, no data are available on the effects of PTH or its fragments on neonatal or very young intact animals. This has prompted us to study and compare the effects of both factors, PTH and PTHrP, on neonatal bones.

In the present study we present results obtained by administering either intact PTH or one of its three synthetic human fragments (1–34, 28–48, or 53–84) and the N-terminal fragment 1–34 of the human PTHrP (hPTHrP) to neonatal mice. We show not only that both N-terminal fragments of PTH and PTHrP have striking anabolic effects on neonatal bones, but also that PTH (28–48) is a prominent mitogenic and anabolic factor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Twenty-two groups of 2-day-old ICR mice were used in this study. Each group (n = 6) was injected sc daily with one of the following peptides: 1) synthetic human PTH (hPTH) (1–84), 2) hPTH (1–34), 3) hPTH (28–48), 4) hPTH (53–84) (all purchased from Sigma Chemical Co., St. Louis, MO), or 5) synthetic hPTHrP (1–34) (Bachem, Torrance, CA). Each peptide was injected daily in two doses, a low dose of 0.05 µg/g body weight (BW) and a higher dose of 0.2 µg/g BW for 6 or 16 consecutive days. Two groups of mice received only vehicle (0.1% BSA in 0.01 M acetic acid) throughout the two experimental periods. The mice were housed under similar conditions, receiving Purina Mouse Chow (Koffolk, Tel-Aviv, Israel) and water ad libitum. They were weighed every day, and the volume of the injected peptide was adjusted to weight gain to maintain the correct dosage. Animals were maintained and killed in a manner approved by the Committee on Animal Care and Use of the Technion Faculty of Medicine.

hPTH (1–84) and its other fragments were dissolved in 0.01 M acetic acid to which 0.1% BSA was added. hPTHrP (1–34) was dissolved in 0.005 M acetic acid that contained 0.5% BSA. Animals were killed by ether anesthesia on either their 8th or 18th day of life, respectively. Tibias, mandibular condyles, and femurs were removed. One tibia, one mandibular condyle, and both femurs from each animal were quickly frozen and stored at -70 C until subsequent biochemical determinations. The second tibia was fixed in 10% neutral buffered formalin and embedded in paraffin.

Biochemical determinations
Tibias and condyles were allowed to reach room temperature, adherent moisture was absorbed, and they were weighed. Individual tibias were homogenized by a Kinematica homogenizer (Kinematika Polytron GmbH, Kriens-Lucern, Switzerland) in a 20-fold volume of buffer (composed of 0.01 M Trisma base (Sigma Chemical Co., St. Louis, MO), 2.5 mM MgCl2, and 0.1% Triton X-100) in three pulses over crushed ice. Condyles were assembled into groups of three for each determination and likewise homogenized. One-hundred-microliter samples were directly analyzed in duplicates for total protein by the method of Lowry et al. (17). Five-hundred-microliter samples were analyzed for their DNA content by the method of Burton (18).

[3H]Thymidine autoradiography
In a separate experiment, two groups of six neonatal mice were injected with hPTH (28–48) at the low dose of 0.05 µg/g BW for 6 and 16 days, and 12 mice received only vehicle for the same time periods. Three hours before death, all mice received a pulse of 6 µCi/g BW of [3H]thymidine (50 Ci/mmol specific activity; Amersham Nuclear Research Centre, Amersham, Bucks, UK). Tibias were removed and fixed in 10% neutral buffered formalin overnight, dehydrated in graded ethanols, and embedded in paraffin. Sections, 5 µm thick, were mounted on glass slides, coated with nuclear track emulsion (NTB-2; Kodak, Rochester, NY), and placed in light-tight boxes at 4 C for 3 weeks. Autoradiographs were developed in Kodak D-170 developer at 18 C, fixed, and lightly stained with hematoxylin and eosin. Sections were photographed at x400 magnification. [3H]Thymidine-labeled cells were counted on the photographs in the following areas: throughout the epiphyseal cartilage of 8-day-old mice and in the proliferation zone of the growth plate of 18-day-old mice. Counts from hPTH (28–48)-injected mice were compared and statistically analyzed with the counts of the vehicle-injected mice.

Tissue insulin-like growth factor I (IGF-I) extraction
Femurs from mice that were injected with the low dose of hPTH (28–48) (0.05 µg/g BW) for 6 or 16 consecutive days were removed and immediately frozen in liquid nitrogen. For IGF-I measurements, frozen femurs were allowed to reach room temperature, then weighed and pulverized under liquid nitrogen using a mortar and pestle. After pulverization, the tissue was weighed and diluted in glacial acetic acid in a ratio of 1:5, vortexed, held on an ice bath for 2 h, then centrifuged at 2400 x g for 30 min, and stored overnight at -70 C. Twenty-four hours thereafter, samples were recentrifuged, and the supernatant was recentrifuged at 15,000 x g. Aliquots were analyzed quantitatively by a rat IGF-I RIA kit (Diagnostic Systems Labs., Webster, TX) following the directions of the manufacturer.

IGF-I in situ hybridization
Rat IGF-I (376 bp) cloned in pGEM3 was used as a probe. After linearization, antisense RNA was transcribed using Sp6/T7 Dig-RNA labeling kit (Boehringer, Mannheim, Germany), following the manufacturer’s instructions. Paraffin sections from tibias were loaded on precleaned polylysine (0.01%)-coated slides. After deparaffination in xylene and hydration in graded ethanols, sections were reacted with 12 mg/ml proteinase K for 15 min at 37 C and acetylated with 0.5% acetic anhydride in 0.1 M Tris, pH 8.0, for 10 min at room temperature. Prehybridization was performed in 2 x SSC for 10 min and 1 h in hybridization buffer containing 50% formamide, 0.5 mg/ml salmon sperm DNA, 4 x SSC, 1 x Denhardt, 5% dextran sulfate, 200 U/ml heparin, and 0.01% SDS. Hybridization was carried out with 2 ng/ml digoxigenin-labeled antisense RNA probe for 18 h at 42 C. Following hybridization, slides were washed in SSC (2x, 1x, and 0.5x), and detection of hybrids was revealed by antidigoxigenin conjugated with peroxidase and aminoethyl carbazole as substrate.

Statistical analysis
All results are the mean ±SD. Differences between means were analyzed using one-way ANOVA. P < 0.05 was assumed to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DNA in tibia
Figure 1Go presents the results of the measurements of DNA content in tibias of 8- or 18-day-old mice that were injected with the two doses (0.05 and 0.2 µg/g BW) of hPTH (1–84) and its three fragments (1–34, 28–48, and 53–84) compared with control, vehicle-injected mice following 6 or 16 daily injections, respectively.



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  		Figure 1. Effect of daily injections of vehicle, hPTH (1–84), and hPTH fragments (0.05 and 0.2 µg/g BW) on DNA content of neonatal mouse tibia following 6 or 16 daily injections.

 
The low doses of hPTH (1–84) and hPTH (1–34) significantly increased DNA after both 6 and 16 daily injections, revealing hPTH (1–34) to be the more effective of the two. The increase of DNA by the higher dose was smaller and was not statistically significant after either interval. On the other hand, the mid-region fragment of PTH, hPTH (28–48), affected DNA when injected at both low and high doses and after both temporal protocols. The carboxyl-terminal fragment of PTH, hPTH (53–84), had no effect on DNA content of the tibia.

Total protein in tibia
Statistically significant increases in total protein content were brought about only by the administration of low doses of hPTH (1–84) and its two fragments hPTH (1–34) and hPTH (28–48), and only following the longer treatment period. No significant increases in total protein were effectuated by the carboxyl-terminal hPTH (53–84) (Fig. 2Go).



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  		Figure 2. Effect of daily injections of vehicle, hPTH (1–84), and hPTH fragments (0.05 and 0.2 µg/g BW) on protein content of neonatal mouse tibia following 6 or 16 daily injections.

 
DNA in mandibular condyle
Figure 3Go presents the results of the measurements of DNA content in mandibular condyles of the different peptide-injected mice. One can see that, unlike in the tibia, in the mandibular condyle a dose-related increase in DNA, especially by the intact hormone and by the mid-region fragment, could be demonstrated. The most effective in increasing DNA was the whole molecule of the hormone. Interestingly, hPTH (1–34) was effective only following 16 injections and only at the higher dose. Thus, the mandibular condyle was not refractive to higher doses of hPTH (1–84) or hPTH (1–34), as was demonstrated by the tibia. The mid-region molecule, hPTH (28–48), was also significantly effective in raising DNA content of mandibular condyles at both injected doses and after both time periods of injections. Similarly to the tibia, the DNA content of mandibular condyles was not changed by the carboxyl-terminal peptide.



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  		Figure 3. Effect of daily injections of vehicle, hPTH (1–84), and hPTH fragments (0.05 and 0.2 µg/g BW) on DNA content of neonatal mouse mandibular condyle following 6 or 16 daily injections.

 
Total protein in mandibular condyles
hPTH (1–84) increased protein in mandibular condyle only when injected at the low dose for 6 days, whereas hPTH (1–34) acted similarly following 6 daily injections with the low dose, and even more effectively with the higher dose, but only when injected for 16 days (Fig. 4Go). The mid-region molecule, hPTH (28–48), was dramatically effective after 6 daily injections. After 16 daily injections, only the higher dose still significantly increased protein content, but to a much lesser extent than after the shorter interval of injections. The low dose of hPTH (28–48), similarly to the other two peptides, did not increase protein significantly after the longer protocol of injections (Fig. 4Go). Again, the carboxyl-terminal had no effect on protein content of the mandibular condyle.



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  		Figure 4. Effect of daily injections of vehicle, hPTH (1–84), and hPTH fragments (0.05 and 0.2 µg/g BW) on protein content of neonatal mouse mandibular condyle following 6 or 16 daily injections.

 
Comparison between effects of hPTH (1–34) and hPTHrP (1–34) on DNA and protein content of tibia
Figure 5Go compares the effects of hPTH (1–34) and hPTHrP (1–34) on the DNA content of the tibia. One can see that hPTHrP (1–34) increased DNA significantly only when administered for 6 days. hPTH (1–34) seemed to be more potent than hPTHrP in this respect, but only when injected at the lower dose, as already presented in Fig. 1Go.



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  		Figure 5. Comparison between effects of hPTH (1–34) and hPTHrP (1–34) (0.05 and 0.02 µg/g BW) on DNA content of neonatal mouse tibia following 6 or 16 daily injections.

 
Although the effect of hPTH (1–34) on DNA was more pronounced than that of hPTHrP (1–34), hPTHrP (1–34) had a remarkably greater effect on total protein at both doses and after both time intervals (Fig. 6Go). hPTH (1–34) increased total protein significantly only at the lower dose and only with the longer treatment protocol.



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  		Figure 6. Comparison between effects of hPTH (1–34) and hPTHrP (1–34) (0.05 and 0.02 µg/g BW) on protein content of neonatal mouse tibia following 6 or 16 daily injections.

 
[3H]Thymidine autoradiography in tibias of hPTH (28–48)-treated mice
[3H]Thymidine autoradiography was used to identify and localize cellular populations engaged in DNA synthesis following stimulation by hPTH (28–48). The proximal epiphysis of the tibia of 8-day-old mice is composed of chondrocytes in different stages of maturation. Some of them are proliferating chondrocytes, as demonstrated by the exhibition of grains in their nuclei in autoradiographs of vehicle-treated mice. Most of them are concentrated in the proliferative zone of the epiphyseal growth plate, which starts to be organized at this age. At this age the secondary center of ossification at the proximal epiphysis has not yet developed. Six consecutive daily injections of hPTH (28–48) brought about a 3-fold increase in the number of labeled cells throughout the epiphyseal cartilage, but mostly in the zone of the future proliferative layer of the growth plate. Following 16 daily injections of hPTH (28–48), a similar increase in the number of labeled cells was observed in the proliferative zone of the growth plate of 18-day-old mice (Table 1Go).


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  		Table 1. Number of [3H]thymidine-labeled cells in epiphyseal cartilage of 8-day-old mice and in epiphyseal growth plate of 18-day-old mice treated with low-dose hPTH (28–48) (0.05 µg/g BW) compared with vehicle-treated mice

 
Remarkably, increased numbers of labeled cells were also observed in the secondary ossification center of the epiphysis of 18-day-old mice following 16 daily injections of hPTH (28–48) and also in the metaphysis of mice that were injected by either the 6- or the 16-day protocol (Fig. 7AGo). These areas could not be counted because of the high uptake of the label by cells in the bone marrow. However, observation of higher magnifications of the autoradiographs could identify the labeled cells in the metaphysis as cells located either directly adjacent to the osteoid surfaces or in close proximity to the invading capillaries, suggesting their identification as pericytes (Fig. 7BGo).



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  		Figure 7. [3H]Thymidine-labeled cells along trabeculae of metaphyseal bone of hPTH (28–48)-treated neonatal mouse. Labeled cells are located along trabeculae (arrowhead) and capillaries (arrow). Magnification: A, x100, bar, 100 µm; B, x400, bar, 10 µm.

 
IGF-I content in femurs of hPTH (28–48)-treated mice
A significant increase (26%) in the amount of IGF-I was noted in the femurs that were removed from hPTH (28–48)-treated mice following 16 injections relative to their vehicle-treated littermates (Table 2Go). IGF-I content in femurs from vehicle-treated mice and from treated mice only for 6 days did not rise above the sensitivity threshold of the detection kit.


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  		Table 2. Measurements of IGF-I content in 18-day-old mice treated with low-dose hPTH (28–48) (0.05 µg/g BW) compared with vehicle-treated mice

 
IGF-I mRNA in situ hybridization
The localization of IGF-I mRNA expression by the in situ hybridization technique was employed in paraffin sections from the proximal tibias of 18-day-old mice. Using this method, sections from mice treated with vehicle for 16 days did not show any IGF-I expression (Fig. 8Go, A, C, and E), nor with an irrelevant digoxigenin-labeled antisense RNA probe (not shown). On the other hand, sections of proximal tibia from hPTH (28–48)-treated mice revealed distinct IGF-I mRNA staining in most of the chondrocytes that occupy the proliferative and early differentiating zones of the growth plate (Fig. 8Go, B and D). IGF-I was also expressed in the population of chondrocytes that reside laterally to the growth plate. These cells contribute to the transverse apposition of cartilage to the bone and are responsible for the enlargement of the epiphysis in the horizontal plane at the level of the growth plate (Fig. 8FGo). In addition, it is possible to appreciate from the same photomicrographs the increase in numbers of the proliferating chondrocytes and of those located laterally to the growth plate in the sections from the hPTH (28–48)-treated mice (Fig. 8Go, B, D, and F) as compared with those from the vehicle-treated mice (Fig. 8Go, A, C, and E).



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  		Figure 8. In situ hybridization of IGF-I mRNA in sections of hPTH (28–48)-treated neonatal mouse proximal tibia. No staining can be seen in vehicle-treated chondrocytes (A, C, and E), whereas positive staining is present in hPTH (28–48)-treated chondrocytes (B, D, and F). In growth plate, mainly cells in proliferative (P) and maturing (M) zones are positive (B and D). A and B, Low-power magnifications (x100, bar, 100 µm); C and D, higher magnifications of proliferating zone of growth plate (x400, bar, 10 µm). Laterally located chondrocytes (L) seen in B are enlarged in E and F (x400, bar, 10 µm).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To the best of our knowledge, our data are the first to demonstrate anabolic effects of PTH and PTHrP fragments in neonatal animals. Although the in vivo anabolic effects of the intact PTH and its N-terminal fragment 1–34 were expected (1, 2, 3, 19), lending support to previous in vitro experiments (15, 16, 20, 21, 22, 23), the remarkable in vivo mitogenic effect of hPTH (28–48) on neonatal bones was unexpected and impressive (24, 25, 26). To date, only one report has dealt with in vivo mitogenic effects of PTH (28–48) (19). Our experiments confirm the importance of a core region functional domain in the mid-region of the PTH molecule for proliferation, suspected to be between amino acids 30 and 34 (27, 28). More precisely, it has recently been reported that some of the mitogenic effects of PTH are believed to be related to amino acids 29–32, which through binding to the common receptor for PTH activates the phospholipase C/protein kinase C signal transduction pathway (29, 30). Furthermore, there are indications for a specific receptor for the PTH (28–48) fragment that also activates protein kinase C (27, 31).

In the present study, the mitogenic effect of all the fragments except the carboxyl-terminal one was high, probably because of the extreme susceptibility of neonatal bones to these peptides. Although the intact hormone and its N-terminal fragment were mitogenic to long bones (tibia) only when administered at the lower dose of 0.05 µg/g BW, hPTH (28–48) was mitogenic even when administered at the higher dose of 0.2 µg BW. Thus, the inhibitory and catabolic effects that are associated with the very first amino acids in the N-terminus of the PTH molecule are excluded from the mid-region fragment (27, 32, 33, 34). As a consequence, the latter could be considered a purely anabolic factor free of catabolic parameters that have traditionally been attributed to PTH.

It has previously been demonstrated that PTH has a biphasic effect. High doses of PTH are inhibitory, whereas lower doses are stimulatory (15, 22). Moreover, because of the stimulating effect that PTH also has on osteoclastic activity, an effect that is believed to be mediated by the osteoblasts and that brings about resorption (35), the net balance between anabolic, inhibitory signals and stimulated resorption could explain why high doses, already following six injections, did not exhibit significant net anabolic effects in a complex organ as a whole bone.

In the present study, DNA was measured in homogenates of whole tibias and in the cartilaginous part of the mandibular condyle. In the tibia, we did not attempt to separate the bone marrow because of the smallness of the specimens. Thus, the tibial homogenates contained cartilage, metaphyseal and diaphyseal bone, and marrow tissue, whereas those of mandibular condyles contained mostly cartilage. It can be argued that the sensitivity of the chondrocytes of the mandibular condyle is less than that of the young long bone. This was especially observed in hPTH (1–34)-treated condyles, in which it exerted an increase in DNA content in the condyle only after the high dose and 16 injections. Because the mandibular condyle is a secondary cartilage that transforms very quickly from functioning as a growth center into an articular cartilage, it might be expected that the density of PTH receptors in its chondrocytes would be different from that in the membranes of epiphyseal chondrocytes and osteoblasts of long bones, and also that their numbers during ontogeny would change differently (36, 37).

In previous in vitro studies by our group, we could not show any mitogenic effect of hPTH (28–48) on the cartilage of the mandibular condyle when grown as an organ-culture system (15). This contradicted other studies in chick and rat chondrocytes’ cell-culture system and in young rats in vivo (19, 24, 25, 26), which did demonstrate such an effect. One explanation could be that the number of receptors expressed in vitro by the chondroprogenitor cells of the condyle is too low to exert an effect, whereas in vivo more favorable conditions prevail. Moreover, the increase in DNA in the condyle in the present study behaved in a dose-related manner in both temporal protocols (Fig. 3Go), which means that the stimulation to proliferation could be maintained also by the somewhat older condyles.

In our previous in vitro study, we observed that the mid-region fragment enhanced maturation, hypertrophy, and mineralization of the chondrocytes (23). This earlier observation is in agreement with the results obtained in the present study, which showed that the proliferative effect of PTH (28–48) was also accompanied by a rise in protein content. Although total protein might not be regarded as a suitable parameter for differentiation, it still is indicative of an overall anabolic effect (Fig. 4Go). In the tibia this effect was not as pronounced and was exerted only by the lower dose following 16 injections. This could be explained by the extreme complexity of the structure of the tibia comprising several different tissues, as noted earlier, when compared with the mandibular condyle, which is mainly cartilaginous.

The increase in IGF-I content and mRNA in the tibias from PTH (28–48)-treated mice suggests that IGF-I might be induced and be responsible, at least in part, for the anabolic effects observed to be exerted by this fragment, as has been previously demonstrated for PTH (1–34) (38, 39, 40).

We were able to demonstrate expression of mRNA for IGF-I in the hPTH (28–48)-treated chondrocytes of the proximal tibia of 18-day-old mice, whereas the control, vehicle-treated mice did not express mRNA for IGF-I. This result correlated well with the increase in content of IGF-I protein in the femurs from the hPTH (28–48)-treated mice and also with the thymidine autoradiography results. Although DNA in the tibia was measured in the whole bone, thymidine autoradiography clearly demonstrated that chondrocytes were targets for PTH (28–48) and responded by stimulated proliferation. Both in the epiphyseal cartilage of 8-day-old mice and in the proliferative zone of the growth plate of the 18-day-old mice, the incidence of labeled cells was increased by 3-fold (Table 1Go). Preosteoblasts might also be targets for the mid-region fragment, because many of their nuclei were labeled by thymidine in the metaphyseal bone of the tibia (Fig. 7Go). This observation is in agreement with the published data about the targets in metaphyseal bone for PTH (41). It was impossible to count their numbers and to compare them with control bones, because of the very high density of label in the adjacent marrow. Still, we are positive that the number of labeled cells not in the marrow, but closer to bone surfaces, was significantly increased. Thus, not only cartilage cells are sensitive to the mid-region fragment, but also preosseous cells.

Of interest are the results of the comparison between hPTH (1–34) and hPTHrP (1–34). Although both proteins are believed to activate the same receptor, they are not identical, sharing about 70% homology (7). In our study, hPTH (1–34) did not increase DNA in the tibia when administered at the higher dose, whereas hPTHrP (1–34) demonstrated a transient mitogenic effect at both doses, i.e. when administered for the shorter period of six injections. Concomitantly, the effect of hPTHrP (1–34) on protein content was maintained following the longer protocol and was exerted by both the lower and the higher dose. Thus, it could be argued that hPTHrP (1–34) brings about a more sustained anabolic effect than hPTH (1–34), an effect that might be brought about by an additional binding site to this peptide that signals for differentiation (7).

In summary, the present study demonstrated that neonatal growth centers are highly susceptible to mitogenic and differentiating effects of PTH fragments and raise the probability of their participation in the regulation of growth during the early stages of life. Moreover, this study revealed the extreme potency in this respect of the mid-region fragment, hPTH (28–48), and the possible involvement of IGF-I as its mediator.


   Acknowledgments
 
We thank Mrs. Cila Shen-Orr for performing the IGF-I RIA and Miss Ruth Singer for her excellent typing and editing of the manuscript.


   Footnotes
 
1 This work was supported in part by Grant 181–910 from the Technion V.P.R. Fund-Loewengart Research Fund. Presented in part at the annual meeting of the Israel Calcified Tissue Society, Petah Tiqva, Israel, March 1994; at the 10th International Workshop on Calcified Tissues, Jerusalem, Israel, March 1996; and at the 3rd Insulin-Like Growth Factors Meeting, Beer Sheva, Israel, March 1996. Back

Received June 20, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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