Differential Effects of Parathyroid Hormone Fragments on Collagen Gene Expression in Chondrocytes
Silke Erdmann, Wolfgang Müller, Safarali Bahrami, Silvia I. Vornehm, Hubert Mayer, Peter Bruckner, Klaus von der Mark, and Harald Burkhardt
Department of Internal Medicine III, Institute for Clinical Immunology and Rheumatology, University of Erlangen-Nürnberg,

Institute for Experimental Medicine, University of Erlangen-Nürnberg,
Institute for Physiology, Section Neurophysiology, Charité, Berlin

Institute for Physiological Chemistry and Pathobiochemistry, University of Münster,
Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, Germany

Materials and Methods

The effect of parathyroid hormone (PTH) in vivo after secretion by the parathyroid gland is mediated by bioactive fragments of the molecule.

To elucidate their possible role in the regulation of cartilage matrix metabolism, the influence of the amino-terminal (NH2-terminal), the central, and the carboxyl-terminal (COOH-terminal) portion of the PTH on collagen gene expression was studied in a serum free cell culture system
of fetal bovine and human chondrocytes. Expression of a1 (I), a1 (II), a1 (III), and a1 (X) mRNA
was investigated by in situ hybridization and quantified by Northern blot analysis. NH2-terminal
and mid-regional fragments containing a core sequence between amino acid residues 28-34 of PTH induced a significant rise in a1 (II) mRNA in proliferating chondrocytes. In addi-tion, the COOHterminal portion (aa 52-84) of the PTH molecule was shown to exert a stimulatory effect on a1 (II) and a1 (X) mRNA expression in chondrocytes from the hypertrophic zone of bovine epiphyseal cartilage.
PTH peptides harboring either the functional domain in the central or COOH-terminal region of PTH can induce cAMP independent Ca2+ signaling in different subsets of chondrocytes as assessed by microfluorometry of Fura-2/AM loaded cells. These results support the hypothesis that different hormonal effects of PTH on cartilage matrix metabolism are exerted by distinct effector domains and depend on the differentiation stage of the target cell.

PARATHYROID hormone plays a predominant role in the regulation of calcium homeostasis by acting mainly on its target tissues in the renal cortex and bone (15, 42).
Soon after secretion the parathyroid hormone (PTH) molecule undergoes rapid proteolysis in the liver resulting in multiple fragments (7). Since most of the calcium regulatory functions could be mapped to the NH2-terminal portion (PTH l-34) of PTH, it was thought that this fragment contains all structural requirements for biological activity of the entire molecule (43, 52).

The other fragments were regarded as inactive metabolites whose functional importance was confined to processing and intracellular transport events during hormone secretion by the cells of the parathyroid gland (PTH 53-84, references 35, 46).
However, there is now increasing evidence for a broader spectrum of target tissues, including cartilage (26, 33, 34), processes (8,13) which are mediated by additional functional domains on the mid-regional (23) and COOHterminal portion (39,40,44) of PTH. For example, PTH (53-
84) increases alkaline phosphatase activity in osteoblastic cell lines (39) and more recent studies showed that PTH (39-84) and PTH (53-84) dose dependently stimulate the differentiation of osteoclast precursors into osteoclast-like cells (25). Moreover, for two domains of PTH their
functional role in the induction of second messenger pathways has been elucidated: the first two NH2-terminal amino acids of PTH are needed for adenylate cyclase Stimulation via the “classical” PTH receptor (24, 43,52), whereas the mid-regional part, aa 28-34, is responsible for
induction of protein kinase C activation in target cells (23).
This domain in the central part of PTH also seems to be critical for the mitogenic effect of the fragments PTH (28- 48) and (1-34) in primary cultures of sternal embryonic chicken chondrocytes (48). A more complex pattern of PTH effects has been demonstrated in a culture System of neonatal murine mandibular condylar explants exposed simultaneously to PTH fragments 1-34, 28-48, and 53-84 (51). Each of these fragments was shown to exert distinct
biological effects (51) on the cartilage morphology, indicating a potential critical role for PTH in normal endochondral ossification.
In contrast to these well characterized PTH effects on cell numbers, cell shape and extracellular matrix morphology, little is known about the ability of the different PTH fragments to induce
quantitative and/or qualitative changes in collagen gene expression by chondrocytes. Therefore, it was the aim of this study to elucidate the regulatory potential of different PTH fragments on collagen metabolism of chondrocytes, which might be a critical underlying mechanism of PTH action on cartilage.

Several studies (8, 13, 30) have indicated that the response of chondrocytes to PTH depends on the source and developmental stage of the cartilage. In embryonic transient cartilage, such as
epiphyseal cartilage of long bone rudiments, chondrocytes rapidly proliferate and undergo a series of differentiation steps.

These stages of chondrocyte differentiation are aligned sequentially from the epiphyseal surface down to the growth zone in the diaphysis and are characterized by the expression of
different collagen types as specific markers (for review see reference 55). Preembryonic cells in the superficial layer express collagen type I, proliferating chondrocytes in the middle
zone synthesize collagen II, VI, IX, and XI, whereas hypertrophic chondrocytes of the growth plate can be unequivocally identified on the basis of their collagen type X expression (16,
49). The expression of this characteristic collagen in the deep zone of the epiphysis seems to be functionally related to endochondral ossification processes (16, 49) in the matrix preceeding cartilage resorption by osteoclasts and replacement by endochondral bone. Since PTH is known to promote endochondral ossification (50), it was the aim of our study to investigate
modulatory effects of PTH fragments on the expression of collagen II and X mRNA in epiphyseal chondrocytes of different developmental stages.

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