the pace of cartilage differentiation. H M Kronenberg, K Lee, B Lanske and G V Segre. Endocrine Unit, Massachusetts General Hospital and Harvard Medical ...
Parathyroid
hormone-related protein and Indian the pace of cartilage differentiation H M
Kronenberg,
K
Lee, B Lanske and G
Endocrine Unit, Massachusetts General
Hospital
(Requests for offprints should be addressed
V
hedgehog
control
Segre
and Harvard Medical School, Boston, Massachusetts 02114, USA
to H M
Kronenberg)
hypercalcemia of malignancy, results from inappropriate use of the renal and bone PTH/PTHrP receptors by PTHrP. In this disease, the normal homeo¬ static mechanisms are disrupted by the secretion of PTHrP by tumors. Unlike the parathyroid cells, the tumor cells do not decrease the secretion of PTHrP when hypercalcemia the humoral
Introduction
Parathyroid hormone (PTH) controls calcium homeostasis through its actions on kidney and bone to raise blood
calcium. Calcium, in turn, suppresses PTH secretion from the parathyroid gland. This negative feed-back loop (Fig. 1) serves to maintain the constancy of blood calcium from minute to minute. The receptor that mediates the actions of PTH is a G-protein-linked receptor that also mediates actions of parathyroid hormone related protein (PTHrP). The normal functions of PTHrP are still incompletely understood, but are likely to include predominantly paracrine actions (Broadus & Stewart 1994). PTHrP is synthesized by many tissues from the earliest stages of development and throughout life, although the blood levels of PTHrP are usually quite low. PTH/PTHrP receptors are often found near the sites of synthesis of PTHrP, and PTHrP has been shown to act on tissues in which PTHrP is synthesized. How one receptor can mediate the calcium homeostatic functions of PTH and, at the same time, mediate the paracrine functions of PTHrP is not clear. One disease,
supervenes. Like most other endocrine diseases, therefore, the humoral hypercalcemia of malignancy illustrates the importance of feedback loops to ensure homeostasis. In this manuscript we shall describe a paracrine feed¬ back loop that controls the local secretion of PTHrP in bone; this feedback loop is formally analogous to the hormonal feedback loop that controls calcium homeostasis. Recent work using gene 'knockout' mice has shown that PTHrP is essential for the normal pace of differentiation of growth plate chondrocytes (Karaplis et al. 1994). PTHrP normally acts to slow the pace of differentiation of these chondrocytes. Chondrocytes that have stopped proliferat¬ ing and have begun to differentiate further start to secrete Indian hedgehog protein (Ihh). Indian hedgehog, through a still incompletely understood pathway, then increases the secretion of PTHrP, which acts directly on chondrocytes
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brain smooth muscle skin
fetal tissues other
PARACRINE ACTIONS
CALCIUM HOMEOSTASIS
Figure 1 Actions of PTH and PTHrP. PTH acts on PTH/PTHrP receptors ¡n kidney and bone to taise blood calcium; calcium supptesses PTH secretion. Together, PTH and calcium act as signals in a negative feedback loop that controls calcium homeostasis. PTHtP, acting predominantly in a paracrine fashion, uses the same PTH/PTHrP receptor to effect a variety of paracrine actions.
Figure 2 A paracrine negative feedback loop controls chondrocyte diffetentiation. Ihh is synthesized by chondrocytes that ate ceasing cell division and switching their genetic program to differentiate into hypertrophie chondrocytes that make type X collagen. The Ihh acts on neatby perichondrial cells. Eithet directly ot indirectly these actions lead to increased synthesis of PTHrP mRNA. The dotted line in the Figute indicates the uncertainty about precisely how far the Indian hedgehog diffuses befóte it acts on perichondrial cells. The simplest possibility is that Ihh acts on perichondrial cells that synthesize PTHrP. Alternatively, the Ihh signal may activate perichondrial cells that do not synthesize PTHrP; this could then trigger a further cascade leading indirectly to increased PTHrP synthesis by other perichondrial cells. The PTHrP then acts to slow down the differentiation of chondrocytes and, theteby, to delay the production of cells that synthesize Ihh. This completes a negative feedback loop. slow the pace of differentiation and, therefore, to slow the production of cells that secrete Ihh (Fig. 2). Thus, a local negative feedback loop ensures that chondrocytes differentiate at an appropriate pace. to
PTHrP controls
growth plate
difFerentiation
Though effects of PTH on isolated growth plate chondrocytes have been noted for many years, the physiologic meaning of these actions was uncertain. The phenotype of mice missing the PTHrP gene, however, strongly suggests that PTHrP, more than circulating PTH, acts during development to control chondrocyte function. Mice missing both copies of the PTHrP gene die at birth with widespread abnormalities in bones formed by endochondral replacement. Throughout the skeleton, chondrocytes stop dividing at an earlier time than normal and differentiate into hypertrophie chondrocytes. This accelerated curtailment of proliferation and early differen¬ tiation are illustrated by the strikingly foreshortened columns of proliferating chondrocytes in the growth regions of long bones and by the premature appearance of hypertrophie chondrocytes even in cartilage that never becomes hypertrophie normally (Fig. 3). Associated
with this accelerated differentiation of chondrocytes is accelerated initiation of osteoblast activity adjacent to the hypertrophie chondrocytes. The osteoid laid down by these osteoblasts leads to the premature appearance of mineralized bone. Many of the resulting bones are misshapen and shorter than normal. The dramatically decreased circumference of the rib cage, for example, may well contribute to the rapid death of these mice in the minutes to hours after birth. During early bone development, PTHrP is synthesized by perichondrial cells closest to the articular surfaces (Lee et al. 1995). The PTH/PTHrP receptor is synthesized in nearby chondrocytes. At first, mRNA encoding the PTH/ PTHrP receptor is expressed diffusely throughout the cartilage mold. As discrete growth regions form, however, the level of PTH/PTHrP receptor is low in proliferating chondrocytes and only reaches high levels in cells making the transition from proliferation to hypertrophy. Thus, the PTH/PTHrP receptor is located where it might well mediate the actions of PTHrP on growth plate differentiation. To test this hypothesis, mice missing the gene encoding the PTH/PTHrP receptor gene were established, again using the gene 'knockout' approach (Lanske et al. 1996). Strikingly, the growth plates of these mice closely resembled the growth plates of the PTHrP
3 Growth plates of normal and PTHrP ( / ) fetal mice expression of Ihh and PTH/PTHrP receptor. The hematoxylin and eosin (H&E)-stained sections from tibias of day
Figure
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16-5 fetal mice contrast the long columns of proliferating chondrocytes in the normal growth plate (A) to the shortened columns in the PTHrP ( / ) growth plate (B). In each photograph, H denotes the hypertrophie chondrocytes immediately below the columns of proliferating chondrocytes. The expression of PTH/PTHrP receptor mRNA in normal (A) and mutant (not shown) mice occurs at the border of proliferating and hypertrophying cells. Ihh expression begins just below the cells that start expressing the PTH/PTHrP receptor mRNA in both normal (A) and mutant (B) mice. Reprinted with permission from Vortkamp et al. (1996). -
'knockout' mice (Fig. 4). Just as in the PTHrP ( / ) mice (that is, mice homozygous for the ablation of the PTHrP gene), in the PTH/PTHrP receptor (-/-) mice widespread accelerated differentiation of chondro¬ cytes and premature mineralization of bone occurred. These genetic experiments provide strong evidence that the PTH/PTHrP receptor mediates the actions of PTHrP —
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chondrocyte differentiation. experiments show that, in vivo, PTHrP is required for normal differentiation of growth cartilage. Other observations emphasize the importance of proper regulation of this action. Patients with Jansen metaphyseal on
These
chondrodystrophy have PTH/PTHrP receptors that are constitutively active; that is, even in the absence of PTH or PTHrP, these receptors are 'turned on' and stimulate the activation of Gs and the formation of cyclic AMP (Schipani et al. 1995). Because of the inappropriate activity
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of the PTH/PTHrP receptors, these patients have hyper¬ calcemia, increased urinary cyclic AMP, and elevated levels of l,25(OH)2D3. These patients also have wide¬ spread growth plate abnormalities. In early life they have rickets-like lesions radiographically, and delays in growth plate mineralization with distorted columns of chondro¬ cytes. Dramatically short stature and a characteristic facies results. The growth plate abnormalities can be viewed as the result of inappropriate PTHrP-like actions, just the blood chemistries mimic those in primary as
hyperparathyroidism.
experiments by Weir et al. (1996) further emphasize the importance of PTHrP in normal development of the growth plate. These workers have overexpressed PTHrP in the growth plates of mice by creating transgenic animals expressing PTHrP down¬ stream of the collagen type II promoter. They have found Recent
Figure 4 Growth plates of normal and PTH/PTHrP receptor ( / ) mice expression of Ihh. The hematoxylin and eosin (H&E)-stained section from humeti of day 18-5 fetal mice demonstrates the similarities of the growth plates from PTHrP ( / ) mice (see Fig. 3) and from PTH/PTHrP receptor ( / ) mice (C). These contrast with the normal growth plate (A). Ihh expression occurs in cells at the same stages of differentiation in normal (B) and mutant mice (D). Reprinted with permission from Lanske et al. (1996). —
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that newborn mice show dramatic slowing of the differ¬ entiation of chondrocytes. This phenotype represents the / ) mouse converse of the abnormality in the PTHrP ( and emphasizes the importance of tight regulation of the PTHrP gene in cartilage: both too little and too much PTHrP cause disease. One might well expect the crucial modulators of PTHrP production to be local. After all, PTHrP acts locally to regulate differentiation of adjacent cells. Consequently, the actions of Ihh, produced locally by maturing and early hypertrophie chondrocytes (Bitgood & McMahon 1995), raised considerable interest. Before describing these actions, it is useful to review the reasons for considering the actions of Ihh in the first place. The hedgehog gene was discovered by Nüsslein-Volhard and Wieshaus (1980) in their Nobel Prize-winning analysis of the genes controlling early development in Drosophila. Hedgehog was shown initially to control polarity of —
—
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in larvae and was subsequently shown to widespread actions in many organs of the fruit fly (Nüsslein-Volhard & Wieschaus 1980, Basier & Struhl 1994). For example, hedgehog controls establishment of the anterior-posterior axes in both the wing and the leg of the developing fruit fly. The hedgehog protein is a secreted protein that acts as a protease to autocatalytically cleave offa biologically active amino-terminal portion, which then acts on nearby cells as a paracrine
segmentation have
factor. Three close relatives of hedgehog sonic hedgehog, Indian hedgehog, and desert hedgehog were found in the chicken and mouse. Most early attention was directed at the functions of sonic hedgehog. Sonic hedgehog was found to be vital for differentiation of neurons and for establishment of the anterior—posterior axis of the vertebrate limb (Johnson & Tabin 1995), for example. -
-
Because Indian
hedgehog
was
expressed
in
developing
5 Actions of PTHrP and sonic hedgehog protein (Shh) on bone expiants from PTHrP ( / ) mice. Hindlimbs from normal (A) or PTHrP ( / ) (B,C) day 16-5 fetal mice were stripped of their skin and cultured in serum-free medium for four days. Type X collagen mRNA was detected by in situ hybridization. Both PTHrP and Shh treatment led to delay in the differentiation to hypertrophie chondrocytes (C) and decrease in cells making type X collagen (A,B). Reprinted with permission from Vortkamp eí al. (1996).
Figure
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cartilage, Vortkamp et al. (1996) isolated the chicken version of Indian hedgehog and explored its function in the chicken limb developmental model. They found that Indian hedgehog was expressed in a discrete portion of the developing growth cartilage at the border of the maturing and hypertrophie chondrocytes. Thus, Ihh was found just distal to and overlapping the cells express¬ ing the PTH/PTHrP receptor gene. Vortkamp et al. (1996) then overexpressed the Indian hedgehog gene by infecting the wing buds of developing chickens with a replication-competent recombinant retrovirus. Uninfected bones developed normally, but adjacent infected bones showed striking abnormalities of cartilage and bone develmouse
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Proliferating chondrocytes failed to differentiate hypertrophie chondrocytes in the infected bones. Vortkamp et al. (1996) did not think that Ihh acted directly on chondrocytes to slow their differentiation, however, since infected bones failed to express the genes, patched and gli, in chondrocytes. These genes have been consistently activated in target cells by hedgehog genes in both Drosophila and vertebrate systems. In the infected chicken wings, patched and gli were widely expressed in the perichondrium and adjacent soft tissues, but not at all in the chondrocytes themselves. Consequently, Vortkamp et al. (1996) suggested that Ihh acts on perichondrial cells indirectly to slow the differentiation of chondrocytes. opment.
into
6 Actions of PTHrP and sonic hedgehog protein on bone expiants from PTH/PTHtP receptor ( / ) mice. Hindlimbs from normal (A,B,C) or PTH/PTHrP receptor ( / ) (D,E,F) day 16-5 fetal mice wete cultured as in Fig. 5 for four days. Type X collagen mRNA was detected by in situ hybridization. With no additions, expression of the type X collagen gene was more widespread in the mutant (D) than in the normal (A) mice. Addition of PTHrP suppressed expression of type X collagen in the normal (B), but had no effect in the mutant (E) mice. Shh also suppressed expression of type X collagen in the notmal (C), but not in the mutant (F) mice. Reprinted with permission from Lanske et al. (1996).
Figure
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The similarity of the actions of Ihh in the chicken limb and of PTHrP in mammalian bones led to collaborative experiments to explore further the functional relationships between these two paracrine factors. First, in the infected chicken wings, PTHrP mRNA was found to be overexpressed in perichondrial cells adjacent to the articular surfaces. To test the functional relationships of Ihh and PTHrP, a murine bone expiant system was characterized by Lee et al. (1995). Whole mouse limbs from mice at day 16-5 of gestation were stripped of their skin and cultured in serum-free medium. In these conditions, normal growth cartilage continues its normal pattern of differen¬ tiation, with generation of hypertrophie chondrocytes over several days (Fig. 5A). Chondrocytes in cultured limbs from PTHrP ( / ) mice (Fig. 5B,C) or from PTH/ PTHrP receptor ( / ) mice (Fig. 6A) also differentiate, -
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although, as expected, the pace rapid than in normal limbs. When PTHrP
(1—34)
was
of differentiation is
added
to
more
cultured normal
limbs, formation of new hypertrophie chondrocytes was completely suppressed (Fig. 5A). This was noted both by traditional histologie criteria, as well as by suppression of
production of a marker of hypertrophie chondrocytes, mRNA encoding collagen type X. As expected, addition of PTHrP (1-34) similarly suppressed the differentiation of chondrocytes from PTHrP ( / ) limbs (Fig. 5B,C) and had
effect when added to PTH/PTHrP receptor ( / ) limbs (Fig. 6D,E). Thus, the normal cultured mouse limbs make enough PTHrP to demonstrate the normal effects of PTHrP on the differentiation of chondro¬ cytes and respond to exogenous PTHrP in an expected fashion. Exogenous PTHrP can reverse the effect of —
—
no —
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ablation of the PTHrP gene and requires the presence of PTH/PTHrP receptors to do so. The mouse limb culture system, therefore, appeared to be a suitable system for testing the actions of Ihh on mammalian bone development. Synthetic Ihh, unfortu¬ nately, was not available at the time of these experiments, so synthetic amino-terminal sonic hedgehog was used instead. The biologically active sequences of Ihh and sonic hedgehog closely resemble each other. Further, Vortkamp et al. (1996) showed that retrovirus expressing sonic hedgehog slowed chondrocyte differentiation in the chicken limb in a way that was identical to the actions of the retrovirus expressing Indian hedgehog. When sonic hedgehog was added to normal mouse bone expiants, differentiation of chondrocytes was blocked in a way that closely resembled the actions of PTHrP (Fig. 5A). Further, sonic hedgehog treatment increased the levels of PTHrP mRNA detected in the perichondrial regions of the limbs through in situ hybridization analysis. In striking contrast, however, sonic hedgehog had no effect when added to limbs from PTHrP ( / ) mice (Fig. 5B,C). Further, like PTHrP, sonic hedgehog had no effect on the limbs from PTH/PTHrP receptor ( / ) —
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mice (Fig. 6F). Thus, mice respond to sonic hedgehog with PTHrP-like action only when the mice have intact PTHrP and PTH/PTHrP receptor genes. We can con¬ clude that the effects of Indian hedgehog revealed in the chicken wing and the mouse limb work through the activation of the PTHrP paracrine system. These observations reveal the actions of a negative feedback loop. Indian hedgehog is made by differentiating chondrocytes that have stopped dividing and have begun the transition to hypertrophie chondrocytes. Indian hedge¬ hog then acts on perichondrial cells. It is not clear how far —
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the Ihh signal can reach. Indian hedgehog might directly stimulate the perichondrial cells that synthesize PTHrP.
Alternatively, Ihh may act only on immediately adjacent perichondrial cells that, in turn, send signals to other perichondrial cells to synthesize PTHrP. Thus, either directly or indirectly, Ihh increases the production of perichondrial PTHrP. In turn, this PTHrP acts on chondrocytes expressing the PTH/PTHrP receptor and slows down the differentiation of chondrocytes and delays the production of cells that synthesize Ihh. In this manner, random fluctuations of production of PTHrP are damp¬ ened to ensure a properly paced rate of differentiation across the growth plate. A molecular and physiological understanding of this feedback loop is just beginning. Many cytokines and
hormones have their
own
independent
actions
on
chondrocyte proliferation and differentiation. Others may well act by modulating the synthesis or actions of PTHrP or Ihh. How many steps and cell types constitute the complete feedback loop need to be determined. One can expect that a number of diseases might involve disruption of this system. Further, it is possible that the still poorly understood paracrine actions of PTHrP in other tissues may well be regulated by local paracrine feedback loops. Since PTHrP is synthesized in many tissues that also synthesize sonic hedgehog, Indian hedgehog, and desert hedgehog proteins, these molecules may interact in other tissues besides bone. Alternatively, a host of distinct regulatory loops may have evolved in different settings. The powerful genetic tools now at hand should provide further clarification of these issues. References Basier K Sc Struhl G 1994 Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature 368 208-214. Bitgood MJ & McMahon AP 1995 Hedgehog and Bmp genes are coexpressed at many diverse sites of cell—cell interaction in the mouse embryo. Developmental Biology 172 126—138. Broadus AE & Stewart AF 1994 Parathyroid hormone-related protein: Structure, processing, and physiological actions. In Tlie Parathyroids. Basic and Clinical Concepts, pp 259—294. Eds JP Bilezikian, MA Levine & R Marcus. New York: Raven Press. Johnson RL & Tabin C 1995 The long and short of hedgehog signaling. Cell 81 313-316. Karaplis AC, Luz A, Glowacki J, Bronson RT, Tybulewicz VLJ, Kronenberg HM & Mulligan RC 1994 Lethal skelatal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes and Development 8 277—289. Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A, Karperien M, Defize LHK, Ho C, Mulligan RC, Abou-Samra A-B, Jüppner H, Segre GV & Kronenberg HM 1996 PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone
growth. Science 273 663—666. Lee K, Deeds JD & Segre GV 1995 Expression of parathyroid hormone-related peptide and its receptor messenger ribonucleic acids during fetal development of rats. Endocrinology 136 453-463. Nüsslein-Volhard C & Wieschaus E 1980 Mutations affecting segment number and polarity in Drosophila. Nature 287 795—801. Schipani E, Kruse Sc Jüppner 1995 A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. Science 268 98-100. Vortkamp A, Lee K, Lanske B, Segre GV, Kronenberg HM Sc Tabin CJ 1996 Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273 613-622. Weir EC, Philbrick WM, Amling M, NefFLA, Baron R & Broadus AE 1996 Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation. Developmental Biology 93 1024.
