PTHrP and PTH/PTHrP receptor expression during development
PTHrP is expressed in the round proliferative chondrocytes (RPCs) of the embryonic growth zone (chondroepiphysis) of the mouse as early as embryonic day 12.5 (E12.5) (1
). Postnatally, the forming secondary ossification center subdivides this population into two subpopulations, one that comes to reside at the top of the chondrocyte columns as the so-called reserve zone and the other that remains in the original subarticular region () (19
). Both PTHrP-expressing subpopulations remain in the mouse indefinitely (11
). The type 1 PTH/PTHrP receptor (PTH1R) is highly expressed in prehypertrophic chondrocytes that lie adjacent to the PTHrP-expressing RPCs throughout the forming endochondral bones ( and ).
Most endochondral bones develop by a process of segmentation of mesenchymal condensations. These segmentations correspond to future joints, and are specified by developmental regulatory molecules from the interzone (22
). PTHrP had not been previously demonstrated to regulate articular chondrocytes, so we began by studying PTHrP and PTH1R expression at forming joint sites. We found PTHrP expression to be confined to these sites rather than to the ends of long bones per se, the latter having been thought previously to be the principal site of PTHrP expression (1
). For example, carpals form via a single ossification center and have as many as six articulations, each of which expresses PTHrP, and at the forming elbow joint β-gal activity is present in the articular regions of the distal humerus and semilunar fossa but not at the olecranon (). In all of these locations, the receptor is expressed in prehypertrophic chondrocytes that lie immediately beneath the PTHrP-expressing articular chondrocytes (). Thus, PTHrP and its receptor are deployed at joint sites coincident with joint specification and always in a pattern in which the PTHrP-expressing cells lie at the articular surface and PTHrP receptor-expressing cells lie immediately subjacent.
If PTHrP were to retard chondrocyte differentiation in articular chondrocytes as it does in growth chondrocytes, this deployment of PTHrP and its receptor would exclude terminally differentiating hypertrophic chondrocytes from the articular cartilage and joint space, preventing their mineralization.
PTHrP regulation of hyaline chondrocytes
The chondrocytes of hyaline cartilage structures must remain undifferentiated in order to function, a phenomenon referred to as “maintenance”. The question posed here was whether PTHrP might regulate hyaline chondrocyte differentiation in a manner similar to its regulation of growth chondrocyte differentiation (1
). There was a lead in this regard based on the fact that the PTHrP-null mouse dies at birth because of pathological mineralization of costal cartilage (14
We carried out proof-of-principle studies of this question in two very simple permanent cartilagenous structures, nasal cartilage and costal cartilage. Both are composed of a tube of hyaline chondrocytes surrounded by a PTHrP-expressing perichondrium (11
, see also ).
In PTHrP-null mice at term, the hyaline chondrocytes of nasal cartilage were fully differentiated into prehypertrophic and hypertrophic chondrocytes, as evidenced histologically as well as by alkaline phosphatase and von Kossa staining (). There was also abundant collagen X and Ihh mRNA expression throughout the PTHrP-null nasal cartilage by ISHH, whereas none was seen in the PTHrP-lacZ or wild-type specimens (data not shown). In the single-allele PTHrP-null mouse, this increase in Ihh expression was reflected in a markedly enhanced periosteal β-gal signal, defining these cells as Ihh target cells (compare ). Exactly the same findings were seen in costal cartilage (data not shown) (13
Thus, these simple hyaline cartilage structures seem to be defended against pathological mineralization solely by the capacity of PTHrP to serve as a brake on hyaline chondrocyte differentiation. That is, PTHrP appears to normally regulate hyaline chondrocyte differentiation in these simple structures via circumferential paracrine signaling from the perichondrium to the PTH1R-bearing hyaline chondrocytes within. Ihh targets the perichondrial cells but not the chondrocytes themselves and therefore has no direct effects in these cells (in contrast to what is described below).
The next question concerned putative PTHrP regulation of articular chondrocytes. In addition to the ancient and embryonic iterations of the chondroepiphyseal growth zones, there exists a lesser-known iteration that occupies one end of the short bones of the hands and feet (8
). Whether these short-bone chondroepiphyses represent a persistence of the primitive structure or evolved secondarily is unclear, but they phenocopy the ancient chondroepiphysis in every detail and might therefore serve as a model system of what a primitive joint structure might have been like. These simple structures contain a single subarticular population of PTHrP-expressing RPCs overlying subjacent populations of mineralizing chondrocytes and bone cells. These cells invade this zone from the primary ossification center via a process referred to as direct ossification (8
The PTHrP-knockout mouse manifests a lethal chondrodysplasia that is driven by promiscuous chondrocyte differentiation and mineralization throughout the endochondral skeleton (1
). The short-bone system in this mouse thus provided an opportunity to see if the chondroepiphysis and/or growth plates might be overrun by this pathological process more or less as seen in nasal and costal cartilage. To maximize the developmental time in which the joints might be at risk, we prolonged parturition to E20.5 with progesterone. Absent PTHrP, we found that both ends of the short bones mineralized prematurely via the direct pathway characteristic of the ancient process and that the secondary ossification centers and growth plates failed to form (). Nevertheless, the residual chondroepiphyseal structures and cartilagenous joint spaces at both ends of the short bones were not invaded by mineralized cells (; see also ).
Thus, in the absence of PTHrP, the joints in short bones were threatened but not broached by mineralizing cells. We also looked at this question in more prototypical joints. In PTHrP-null mice, we observed that Ihh-expressing prehypertrophic chondrocytes and collagen X-expressing hypertrophic chondrocytes approached the joints at the elbow and in the digits but here too did not invade the joint space itself (). This same pattern was seen histologically in the form of hypertrophic cells that approached to within several cell layers of the joint in the PTHrP-null as compared to the PTHrP-lacZ specimen ().
Closer inspection of the PTHrP-null histological sections revealed that the surface articular chondrocyte population did not have the spindle-shaped morphology that typifies the normal lining cells () but rather the prototypical appearance of RPCs (). Further, this RPC-like population appeared to be expanded as compared to that in the PTHrP-lacZ mouse, and the marked β-gal overexpression in the single-allele system clearly identified these RPC-like cells as Ihh target cells ().
Ihh is mitogenic for growth RPCs, whereas PTHrP maintains these cells in the cell cycle but is not itself a mitogen (1
). Were Ihh to have the same mitogenic effect in young articular chondrocytes that it has in growth RPCs, it might be responsible for the dramatically different findings seen in articular versus costal/nasal cartilage in the PTHrP-null mice. We looked at this question by examining BrdU incorporation into the nuclei of chondrocytes in wild-type and single-allele PTHrP-null mice at E15.5 and 16.5, reasoning that such proliferation would be apparent several days in developmental time ahead of the established pattern seen at term (). In the wild-type specimens, we found a high rate of BrdU incorporation scattered throughout the developing carpals and digits, as expected at this age (), whereas in the PTHrP-null specimens a high rate of BrdU incorporation was confined to the chondrocytes at the articular margins of the MP joints and carpals (, respectively). This highly anatomical pattern of proliferation reflected premature chondrocyte differentiation in the PTHrP-null mice, as both structures exhibited a promiscuous advance of Ihh-expressing prehypertrophic chondrocytes to positions just subjacent to the articular margins (). Because of its anatomical simplicity, the central carpal bone lent itself to easy quantification on this regard (see legend ).
Thus, the Ihh-PTHrP feedback system appears to be fully deployed in young articular chondrocytes, and this regulatory system would seem to be particularly adept at protecting the forming joints in a combinatorial sense, as Ihh drives proliferation of undifferentiated chondrocytes flanking the joint, while PTHrP normally serves to prevent these cells from exiting the cell cycle.
PTHrP is expressed in the load-bearing articular surfaces of the synovial joints from embryonic life through adulthood. The PTHrP gene is known to be mechanically-induced in a number of sites (12
), so that this pattern suggested that mechanical loading might regulate PTHrP expression in articular cartilage as well. Since weight-bearing joints presumably would be loaded at baseline, we employed unloading techniques to look at this question. The femoropatellar joint was an attractive target because it is particularly heavily loaded in rodents that walk on a flexed knee (28
) and because it is so simply unloaded by transecting the patellar ligament. We also unloaded the knee in separate experiments; since this method may have introduced a degree of joint instability as well, only short-term results are described here.
In femoropatellar surfaces unloaded for 7 days, there was a marked diminution in β-gal-expressing mid-zone chondrocytes, accompanied by an equally marked increase in subjacent alkaline phosphatase-expressing chondrocytes in the deep zone (). These changes were quantified by histomorphometry (see legend) We saw the same changes in the tibial plateaus of knees unloaded for three days () as well as for as few as 24 hrs (not shown). The degree and the rapidity of the articular chondrocyte differentiation in the deep zone was corroborated by the expression of collagen X mRNA (). While the differentiating articular chondrocytes appeared to approximate the articular surface in the unloaded joints (), in none of the specimens did these cells broach the surface, nor did we see any evidence of chondrocyte degeneration. Thus, the unloading-associated changes in PTHrP expression and chondrocyte differentiation in articular cartilage seemed to be very rapid, were not progressive, and did not lead to degenerative findings. Since it is known that physiological physical forces are anabolic rather than catabolic for articular cartilage (29
), our experimental findings very likely reflected the effects of an absence of physiological loading.
Ihh has also been reported to be load-induced (31
), suggesting that unloading might reduce Ihh expression as well as that of PTHrP. On the other hand, the findings just described (the unloading-induced decrease in PTHrP expression and the increase in differentiating and likely Ihh-expressing chondrocytes) would argue for an unloading-associated increase rather than a decrease in Ihh expression. This point was examined by ISHH in both the unloaded patella and knee, each of which demonstrated a clear-cut increase in Ihh expression. This response was seen as early as three days of unloading and persisted for up to four weeks ().Thus, the PTHrP-Ihh regulatory system and its effect on chondrocyte differentiation clearly trumps any potential independent mechanical effect on Ihh expression in articular cartilage. The unusual aspect of Ihh signaling in these experiments was the absence of PTHrP expression in the face of the marked increase in Ihh expression, representing an uncoupling of the Ihh-PTHrP expression pattern that typifies regulation in growth chondrocytes (1