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Discussion

• Abstract
• Materials and Methods
• Results
• References

The results presented in this study show that different fragments of the PTH molecule stimulate collagen type II and X gene expression in chondrocytes under serum-free conditions. In initial experiments using postnatal human costal chondrocytes, PTH (1-34) and PTH (52-84) stimulated synthesis of collagen type II and X in a serum-free agarose culture System. Due to the restricted availability of appropriate amounts of human chondrocytes and the disadvantages of the agarose culture System for studies of gene expression at the mRNA level, further experimentation was performed using a serum-free culture System of bovine growth plate chondrocytes, separated into cells from the resting and proliferating zone, and in cells from the hypertrophic zone.

In agreement with the Stimulation of collagen type II and X at the protein level, a rise in a1 (II) and a1 (X) mRNA level was detected in response to different PTH petides. The results clearly show that the stimulatory PTH effect on collagen mRNA levels is dependent on the differentiation stage of the cells and induced by at least two different functional domains of PTH.

The first domain is located in the central part of the PTH molecule between aa 28-34 and is capable of stimulating a1 (II) expression in resting and proliferating fetal chondrocytes.
A second domain is located in the COOH-terminal part of PTH between aa 52-84.

This domain is recognized only by cells which are differentiating towards the hypertrophic stage; it is not active on proliferating chondrocytes.
Fragments lacking the genuine NH2-terminus (aa residues 1-3) of the hormone, which is indispensible for activation of the PTH receptor associated adenylate cyclase (15, 43, 52) also stimulate type II collagen expression.

This indicates that cAMP does not play a critical role in the signaling pathway of PTH-mediated upregulation of type II collagen gene expression.
All functionally active fragments are capable of inducing a rise in calcium concentration in the chondrocytes as shown in Fig. 9. Similarly, a cAMP independent Ca2+- signaling, involving protein kinase-C activation (57) has been demonstrated in other experimental Systems of PTH action (48). The activation of this signal transfer cascade by PTH is dependent on a region (aa 28-34) in the central part of PTH (23). We mapped the functional domain for Stimulation of collagen type II gene expression in proliferating chondrocytes to the same region. It is, therefore, very likely that a Ca2+ signal induced by the protein kinase-C domain of PTH (23) is also involved in the upregulation of a1 (II) expression in proliferating chondrocytes.

Figure 9. Ratio imaging of intracellular free calcium in bovine fetal chondrocytes as determined by Fura-2 fluorescence. Monolayer cultures of chondrocytes from the hypertrophic zone were stimutated by a bolus application (final concentration: 10-8 M) of PTH (52-84)

(A2 PTH5 and B6 PTH5), PTH (28-48) (A4 PTH2 and B2 PTH2), or PTH (1-34) (B4 PTH1).
A shows representative ratio Images obtained by a Stimulation experiment with a sequential application of PTH peptides 52-84 and 28-48. A1 is the control image before Stimulation with PTH (52-84). A3 shows the return to a stable baseline level and represents the control image preceding Stimulation with PTH (28-48). B shows the images derived from a sequential Stimulation experiment with three different PTH fragments: PTH (28-48) (B2 PTH2), PTH (1-34) (B4 PTH1), and PTH (52-84) (B6 PTH5). B1 Cont is the control image before Stimulation; B3 Cont and B5 Cont demonstrate the return to stable baseline after the preceeding stimulations with the respective PTH peptides. Bar, 20 mm.

In chick chondrocytes, it has been shown (48) that this central PTH domain mediates an EGTA-sensitive mitogenic effect on the cells. However, under the experimental conditions of this study which are high plating density of the cells (1.3-1.91 05/cm2), a 24 h period of hormone treatment, and strict serum-free conditions, no mitogenic effect was detectable for any PTH
fragment.

These culture conditions account for the absence of any proliferative response of the chondrocytes to PTH, which is in accordance with the observations made by Schlüter et al. (48) that high cell densities impede the mitogenic effect of PTH. Therefore, the same functional domain of PTH (amino acid residue 28-34) can exert quite different effects, either a mitogenic response or an increase in differentiated function (a1 (II) expression), depending on the duration of the hormonal Stimulus and the cell density.
Table I. Ca2+ Response Frequency in Fetal Bovine Chondrocyte from the Hypertrophic Zone of the Epiphysis: Ratio Image Analysis PTH (1-34) PTH (28-48) PTH (52-84) FCS Responses (n) 19 24 35 127 Total cells (n) 50 85 119 150 Response 26.0 28.2 29.4 84.6

Fura-2 loaded chondrocytes from the hypertrophic zone of cartilage were examined by microfluorometry. Changes of intracellular free Ca2+ were determined in individual cells by ratio image analysis.

A response was considered to have occured when at least an increase of 0.5 mM from the
baseline Ca2+ was recorded. The number (n) of responsive cells upon Stimulation by different PTH peptides and with FCS is shown.

The response frequency was calculated from the ratio of responsive cells to total cells measured.

Figure 9. Ratio imaging of intracellular free calcium in bovine fetal chondrocytes as determined by Fura-2 fluorescence. Monolayer cultures of chondrocytes from the hypertrophic zone were stimutated by a bolus application (final concentration: 10-8 M) of PTH (52-84)
(A2 PTH5 and B6 PTH5), PTH (28-48) (A4 PTH2 and B2 PTH2), or PTH (1-34) (B4 PTH1). A shows representative ratio Images obtained by a Stimulation experiment with a sequential application of PTH peptides 52-84 and 28-48. A1 is the control image before Stimulation with PTH (52-84). A3 shows the return to a stable baseline level and represents the control image preceding Stimulation with PTH (28-48). B shows the images derived from a sequential Stimulation experiment with three different PTH fragments: PTH (28-48) (B2 PTH2), PTH (1-34) (B4 PTH1), and PTH (52-84) (B6 PTH5). B1 Cont is the control image before Stimulation; B3 Cont and B5 Cont demonstrate the return to stable baseline after the preceeding stimulations with the respective PTH peptides. Bar, 20 mm.

These environmental influences are critical for PTHmediated effects, hence differences in culture conditions may also account for some apparently controversial results found in the literature on the hormone action on Collagen synthesis by chondrocytes (13, 21, 36, 41). In the study of
Crabb et al. (13) articular chick chondrocytes remained totally unresponsive to PTH, whereas a mitogenic effect and Inhibition of collagen synthesis was reported in growth plate chondrocytes. Similar inhibitory effects of PTH (1- 34) and PTH (54-84) on collagen X synthesis by
hypertrophic rabbit chondrocytes were recently reported by Iwamoto et al. (21).

In a detailed analysis of PTH effects on long term cultures of maturing chick sternal and tibial growth plate chondrocytes, Iwamoto et al. (21) demonstrated that PTH has an inhibitory effect on the emergence of collagen X expressing cells and that this inhibitory effect persisted for the whole maturation pathway of the chondrocytes, which is in contrast to the more stage-specific effects of FGF-2 (22).
However, these experiments were performed in long term cultures and in the presence of 5-10% FCS in the culture medium, while all stimulatory effects of the PTH fragments on collagen gene expression reported here depend on strict serum-free conditions. We have shown that PCS dramatically enhances collagen gene expression within 24 h.

As uncontrolled effects by endogenous growth factors in PCS cannot be excluded (10), serum was omitted from all stages of the experiments reported here. In agreement with Iwamoto et al. (21, 22) we found in our culture System that the stimulatory PTH (1-34) effect on collagen gene
expression was not only abolished, but also reverted, when the chondrocytes were exposed to serum during collagenase digestion before the PTH Stimulus (Vornehm, S., manuscript in preparation). Reduced viability of the chondrocytes prepared and cultured under serum-free conditions was excluded by the fact that freshly prepared cells showed strong signals for a1 (II) mRNA and divided normally.
Since prolonged enzymatic digestion of cartilage in the absence of serum might cause cell damage, care was taken to reduce the time of enzyme treatment to a minimum.

Properly treated chondrocytes remained viable and retained their FCS-responsiveness, as well as responsive-ness to PTH. This PTH response of freshly isolated cells remained stable for a culture period of at least 3d under strict serumfree conditions, however, preference was given in this study to an immediate Stimulation of the chondrocytes already 6 h after Isolation in order to exclude any uncontrolled influence of in vitro (de)differentiation.

The stimulatory effect of PTH peptides on collagen types II and X expression is not restricted to mRNA levels and monolayer conditions; identical results were obtained at protein level with human costal chondrocytes cultured in agarose Suspension. Thus, it is likely that the apparent conflict between our data and those published by Iwamoto et al. (21, 22) result from different chondrocyte culture Systems and reflect the biologically relevant sensitivity of PTH effects to modulation by growth factors present in serum.
Analysis of serum factors that modify the PTH effect would help in the understanding of the complex regulation of collagen metabolism during endochondral bone formation.
In this study a new effector domain for chondrocytes was localized in the COOH-terminal region of PTH (amino acid residues 52-84), which exerts a selective effect on collagen type II and X expression in growth plate chondrocytes from the hypertrophic zone. Proliferating chondrocytes did not respond to PTH peptides derived from the COOH terminus.
It has been shown by Murray et al. (40) that cells differentiated towards the osteoblastic lineage (human osteosarcoma SaOS-2 cells) increase type I collagen mRNA levels in response to PTH (1-34), but not to PTH (53-84), although the COOH-terminal fragment stimulated expression of mRNA for osteocalcin, the vitamin Dreceptor and alkaline phosphatase in the same cells.

This underlines the domain specificity and differentiation stagedependency of the PTH action and supports the concept of a physiological role for PTH metabolites in the hormonal control of matrix metabolism in the growth plate. In accordance with this hypothesis are results from in situ
hybridization studies on fetal rat cartilage (32), showing strong PTH receptor gene expression in a distinct zone of maturing chondrocytes immediately above the layers of hypertrophic cartilage. Moreover, by light microscope autoradiography, Barling and Bibby (5) demonstrated
[3H]PTH binding to hypertrophic chondrocytes; a histological study from 1943 revealed hypertrophy, calcification, and premature closure of the growth plate induced by intraperitoneal administration of PTH to growing mice (50). In an organ culture system of mandibular explants, the COOH-terminal fragment PTH 53-84 exerted a profound change of morphology in the zone of hypertrophic cartilage (51).
In this respect our results suggest that one important facet of PTH action is the Stimulation of collagen type X gene expression in the hypertrophic zone of the epiphysis.
Moreover, modulation of collagen metabolism could be a critical event in calcification of growth plate cartilage since recent data (27, 28, 29) indicate that the interaction of collagen type II and X with the matrix vesicles in the growth plate activate Ca2+ loading of these extracellular microstructures, which are considered the Initiation sites of mineral deposition in cartilage.
Another aspect of the role of PTH in cartilage mineralization is closely related to our finding that PTH metabolites are capable of inducing a rise in intracellular free Ca2+ in chondrocytes from the hypertrophic zone.
Matrix vesicles are formed in chondrocytes by budding from the cytoplasmatic membrane (3, 19) leaving the possibility open that they retain the chondrocytic PTH receptors in their cell membrane. For mineralization of cartilage, it remains to be elucidated whether the matrix
vesicles still respond to PTH fragments after deposition in the extracellular matrix by increasing the intravesicular Ca2+ concentration.
It is not yet clear how the COOH-terminal part of PTH is recognized by the hypertrophic chondrocytes, and why the proliferating chondrocytes remain unresponsive to COOHterminal PTH fragments. PTH and PTH-related peptide (PTHrP) bind to a common heptahelical G-protein coupled receptor molecule (24, 47). Since this classical PTH receptor has a widespread tissue distribution, receptor heterogeneity as a consequence of alternative splicing of the intron-rich PTH receptor gene (23, 31) is an attractive hypothesis for explanation of the observed heterogeneous
PTH responses in the chondrocyte. However, direct proof for the existence of such receptor isoforms in cartilage is lacking. A more ligand selective isoform of the PTH/PTHrP receptor has been identified and characterized for its unresponsiveness to PTH-related peptide (PTHrP), but this PTH 2 receptor seems to be particularly abundant in pancreas and brain and also recognizes the aminoterminal fragment of PTH (54). However, more recently a novel PTH receptor with specificity for the carboxylterminal region of PTH has been characterized in rat osteosarcoma and parathyroid cell lines (20). Moreover, in osteosarcoma cell lines (ROS 17/2.8), this COOH-terminal receptor seemed to be upregulated in response to PTH
Stimuli (PTH 1-34) that are mediated via the common PTH/PTHrP receptor, implying its role in C-receptor expression (20).
Thus, the possibility remains that one of the above mentioned receptors may be involved in the stimulatory effect of the COOH-terminal part of PTH. Receptor isoforms could also explain the differential effects of central vs COOH-terminal PTH peptides on the induction of Ca2+ signaling in distinct cell subsets. Alternatively, Civitelli et al. (11) suggested a nonuniform distribution of functional receptors over the cell surface to explain similar heterogeneous calcium responses to PTH in the osteogenic sarcoma cell line UMR 106. A conformational change in a common receptor molecule could also account for the selective action of the different functional domains of PTH on distinct subpopulation of chondrocytes. Irrespective ofthe receptor molecule involved, our results suggest that two distinct functional domains on the PTH molecule can
exert different hormonal effects on collagen II and X metabolism by epiphyseal chondrocytes, depending on the differentiation stage of the cells.
We thank Dr. G. Gross (GBF Braunschweig) for helpful discussions and Dr. L. Sorokin for reading the manuscript. Initial Ca2+-imaging experiments were performed in the laboratory of Professor Dr. D.
Swandulla (Department of Pharmacology and Toxicology, University of Erlangen). We are grateful to Professor Dr. J.R. Kalden (Department of Internal Medicine III, University of Erlangen) for generous support.
This work was supported by the German Ministry of Research and Technology (BMBF No. 01VM8702) and by the Deutsche Forschungsgemeinschaft (Heisenberg grant to Wolfgang Müller, grant Br 1497/1-1 to Peter Bruckner, and SFB263 grant C3 to H. Burkhardt).
Received for publication 9 July 1996 and in revised form 29 August 1996.

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