Strontium as a tracer to study the transport of calcium in the epiphyseal growth plate (electronprobe microanalysis)

At the mineralization front of the epiphyseal growth plate large quantities of calcium (Ca) are necessary to form the mineral (a Ca-phosphate). It is an unsolved problem, whether Ca is transported through the cells of the growth plate cartilage or extracellularly. Electronprobe microanalysis (EPMA) allows the quantitative, morphologically correlated analysis of elements. EPMA can discriminate only different elements. To investigate the transport of Ca, Strontium (Sr) is a very good tracer, as it resembles Ca in many biological reactions. Our results demonstrate that the transport of Sr from the blood into the growth plate and through the growth plate needs only one or a few minutes. The measured intracellular Sr and Ca concentrations are much lower than the extracellular ones, while the intracellular Sr/Ca ratio is not or only a little bit lower than the extracellular one. It must be concluded, that significant amounts of Ca are neither transported through nor accumulated in the cells of the growth plate cartilage. The main transport is an extracellular diffusion. Using Sr as a tracer for Ca new results on the behaviour of Ca could be received.     

Collagen/annexin V interactions regulate chondrocyte mineralization

Physiological mineralization in growth plate cartilage is highly regulated and restricted to terminally differentiated chondrocytes. Because mineralization occurs in the extracellular matrix, we asked whether major extracellular matrix components (collagens) of growth plate cartilage are directly involved in regulating the mineralization process. Our findings show that types II and X collagen interacted with cell surface-expressed annexin V. These interactions led to a stimulation of annexin V-mediated Ca(2+) influx resulting in an increased intracellular Ca(2+) concentration, [Ca(2+)](i), and ultimately increased alkaline phosphatase activity and mineralization of growth plate chondrocytes. Consequently, stimulation of these interactions (ascorbate to stimulate collagen synthesis, culturing cells on type II collagen-coated dishes, or overexpression of full-length annexin V) resulted in increase of [Ca(2+)](i), alkaline phosphatase activity, and mineralization of growth plate chondrocytes, whereas inhibition of these interactions (3,4-dehydro-l-proline to inhibit collagen secretion, K-201, a specific annexin channel blocker, overexpression of N terminus-deleted mutant annexin V that does not bind to type II collagen and shows reduced Ca(2+) channel activities) decreased [Ca(2+)](i), alkaline phosphatase activity, and mineralization. In conclusion, the interactions between collagen and annexin V regulate mineralization of growth plate cartilage. Because annexin V is up-regulated during pathological mineralization events of articular cartilage, it is possible that these interactions also regulate pathological mineralization.     

Role of the progressive ankylosis gene (ank) in cartilage mineralization

Mineralization of growth plate cartilage is a critical event during endochondral bone formation, which allows replacement of cartilage by bone. Ankylosis protein (Ank), which transports intracellular inorganic pyrophosphate (PP(i)) to the extracellular milieu, is expressed by hypertrophic and, especially highly, by terminally differentiated mineralizing growth plate chondrocytes. Blocking Ank transport activity or ank expression in terminally differentiated mineralizing growth plate chondrocytes led to increases of intra- and extracellular PP(i) concentrations, decreases of alkaline phosphatase (APase) expression and activity, and inhibition of mineralization, whereas treatment of these cells with the APase inhibitor levamisole led to an increase of extracellular PP(i) concentration and inhibition of mineralization. Ank-overexpressing hypertrophic nonmineralizing growth plate chondrocytes showed decreased intra- and extracellular PP(i) levels; increased mineralization-related gene expression of APase, type I collagen, and osteocalcin; increased APase activity; and mineralization. Treatment of Ank-expressing growth plate chondrocytes with a phosphate transport blocker (phosphonoformic acid [PFA]) inhibited uptake of inorganic phosphate (P(i)) and gene expression of the type III Na(+)/P(i) cotransporters Pit-1 and Pit-2. Furthermore, PFA or levamisole treatment of Ank-overexpressing hypertrophic chondrocytes inhibited APase expression and activity and subsequent mineralization. In conclusion, increased Ank activity results in elevated intracellular PP(i) transport to the extracellular milieu, initial hydrolysis of PP(i) to P(i), P(i)-mediated upregulation of APase gene expression and activity, further hydrolysis and removal of the mineralization inhibitor PP(i), and subsequent mineralization.     

Immunohistochemical localization of alkaline phosphatase in the chicken epiphyseal growth cartilage

Summary Alkaline phosphatase of chicken epiphyseal cartilage has been localized by two immunohistochemical methods. Double layer immunofluorescence and peroxidase anti-peroxidase (PAP) methods gave similar results. Alkaline phosphatase in epiphyseal cartilage is extracellular as well as intracellular in the localization. Extracellular reaction was strongest in the lower layers of growth plate and the most intense reaction was noted in the pericellular lacunae of hypertrophic chondrocytes. Also intracellular immunoreaction was noticed through the whole growth plate.     

Zur Resorption von Calcium und Strontium im Dünndarm von Ratte,Maus und Meerschweinchen

The possible modes of permeation of Ca and Sr through the intestinal epithelium are discussed. The problems of the diffusion and the active transport were studied with prepared bags of duodenum, jejunum and ileum of normal but still immature animals (both sexes) in the Warburg apparatus, at body temperature and in some cases at 4° C. Active transport occurs under oxygen, whereas under nitrogen and blockade of the metabolism with cyanide, only diffusion takes place. Isotopes (⁴⁵Ca and⁹⁰Sr) of high specific activity were used. The diffusion of both elements shows the same ratio at low and at body temperature, with an higher intensity of course at 38° C. This ratio (Ca/Sr) is approximately 3 in the duodenum and in the proximal parts of the jejunum; in the distal parts of the intestine, it is approximately 2 or lower. During active transport there is no alteration in these ratios within the serosa in the tissue. Therefore the selection between the two elements takes place immediatly at the entrance in the mucosa. There are only small and negligible differences between rats and mice. In the intestine of guinea pigs, however, a remarkably higher permeation of Ca and lower retention in the tissue, and consequently higher quotients for Ca/Sr, can be observed. The application of Ca-salts (gluconate, lactate) enhances the permeation of Ca and diminishes the entrance of Sr in all animals. The fact that this alteration takes place during conditions of diffusion as well as of active transport supports the assumption that the selection is preferably dependent upon the differences of the physical state of the two ions (atom volume, hydratation of the ions) rather than on their chemical properties.     

The calcification of cartilage matrix in chondrocyte culture: studies of the C-propeptide of type II collagen (chondrocalcin)

We have shown that when chondrocytes are isolated by collagenase digestion of hyaline cartilage from growth plate, nasal, and epiphyseal cartilages of bovine fetuses they rapidly elaborate an extracellular matrix in culture. Only growth plate chondrocytes can calcify this matrix as ascertained by incorporation of 45Ca2+, detection of mineral with von Kossa's stain and electron microscopy. There is an extremely close direct correlation between 45Ca2+ incorporation in the first 24 h of culture and the content of the C-propeptide of type II collagen, measured by radioimmunoassay, at the time of isolation and during culture. Moreover, growth plate cells have an increased intracellular content of the C-propeptide per deoxyribonucleic acid and, during culture, per hydroxyproline (as a measure of helical collagen) compared with nasal and epiphyseal chondrocytes. In growth plate chondrocytes 24,25-dihydroxycholecalciferol (24,25-[OH]2D3), but not 1,25-dihydroxycholecalciferol alone, stimulates the net synthesis of the C-propeptide and calcification; proteoglycan net synthesis is unaffected. Together, these metabolites of vitamin D further stimulate C-propeptide net synthesis but do not further increase calcification stimulated by 24,25-(OH)2D3. These observations further demonstrate the close correlation between the C-propeptide of type II collagen and the calcification of cartilage matrix.     

Relation Between Simultaneous Ca and Sr Transport Rates in Isolated Segments of Vetch, Barley, and Pine Roots

Root segments of vetch, barley, and pine were exposed to a nutrient solution containing (85)Sr and (45)Ca tracers. Translocation was measured from solutions containing stable ions at concentrations of 2.5 mm Ca, and at either 0.5 mm or 2.5 mm Sr. Polar transport was established between 12 and 18 hr in barley, and between 16 and 22 hr in vetch. Acropetal transport remained below 5% of basipetal transport of tracer during these intervals. Transport in both vetch and barley usually declined before an elapsed time of 24 hr unlike corn, which maintained its steady state beyond 24 hr. Pine was radically different in that it showed no difference between acropetal and basipetal transport rates and had very low rates. Sr transport in all plants studied to date paralleled that of Ca and the ratio Sr:Ca transported was equal to the ratio Sr:Ca in the nutrient. In vetch, stable Ca transport was reduced to one-fifth when Sr concentration was increased from 0.5 mm to 2.5 mm. Yet stable Sr transport did not change, indicating that the effect on transport was not due to competitive inhibition. A similar effect was less pronounced in barley, but could not be detected in pine. The magnitude of the transport rates varied considerably among the various species, corn having the greatest followed by barley, vetch, and pine in decreasing order. Transport did not correlate with root weight or surface area; it amounted to from 0.03 to 0.60 nanomoles per hr in these experiments as compared to 7 nanomoles per hr previously established in corn (in all cases, 55 mm segments, sectioned 10 mm from apex).     

Solute transport in growth plate cartilage: in vitro and in vivo

Bone elongation originates from cartilaginous discs (growth plates) at both ends of a growing bone. Here chondrocytes proliferate and subsequently enlarge (hypertrophy), laying down a matrix that serves as the scaffolding for subsequent bone matrix deposition. Because cartilage is generally avascular, all nutrients, oxygen, signaling molecules, and waste must be transported relatively long distances through the tissue for it to survive and function. Here we examine the transport properties of growth plate cartilage. Ex vivo, fluorescence photobleaching recovery methods are used in tissue explants. In vivo, multiphoton microscopy is used to image through an intact perichondrium and into the cartilage of anesthetized mice. Systemically introduced fluorescent tracers are monitored directly as they move from the vasculature into the cartilage. We demonstrate the existence of a relatively permissive region at the midplane of the growth plate, where chondrocytes transition from late proliferative to early hypertrophic stages and where paracrine communication is known to occur between chondrocytes and cells in the surrounding perichondrium. Transport in the living mouse is also significantly affected by fluid flow from the two chondro-osseus junctions, presumably resulting from a pressure difference between the bone vasculature and the cartilage.     

Relation Between Calcium and Strontium Transport Rates as Determined Simultaneously in the Primary Root of Zea mays

Root segments of Zea mays 55 mm long, were exposed to nutrient containing ⁸⁵Sr and ⁴⁵Ca tracers. Translocation rather than uptake was measured, using a newly-designed glass compartmentation system and validated tracer analytic model. Ca transport from solutions containing between 0.25 and 5.0 mm Ca was only slightly affected by concentration, but translocation from 0.25 to 0.05 mm solutions was markedly reduced. Maximum transport of strontium from nutrient containing 0.05 mm Ca was twice that from 2.5 mm Ca, and also twice the maximum calcium transported. Thus, under the condition simulating calcium depletion, i.e., 0.05 mm Ca, greater amounts of strontium were transported. In these cases the solutions also contained stable strontium at concentrations between 0.25 and 5.0 mm. In simultaneous determinations, the ratio of Sr to Ca moved was exactly equal to the ratio of their concentrations in nutrient solution, and there was no evidence of discrimination. Dinitrophenol reduced transport of Sr and Ca to an equivalent extent, amounting to between 2 and 9% of non-treated control levels.     

Uptake and intracellular sequestration of divalent cations in resting and methacholine-stimulated mouse lacrimal acinar cells. Dissociation by Sr2+ and Ba2+ of agonist-stimulated divalent cation entry from the refilling of the agonist-sensitive intracellular pool

The abilities of various divalent cations to enter the cytoplasm of mouse lacrimal acinar cells was examined under resting and agonist-stimulated conditions, by monitoring their effects on the fluorescence of cytosolic fura-2. In vitro, Ni2+, Co2+, and Mn2+ quenched the fura-2 fluorescence, whereas Sr2+, Ba2+, and La3+ produced an excitation spectrum and maximum brightness similar to Ca2+. Stimulation of mouse lacrimal acinar cells with methacholine (MeCh) caused a biphasic elevation of intracellular Ca2+ concentration [( Ca2+]i) resulting from a release of Ca2+ from intracellular pools followed by a sustained entry of extracellular Ca2+. Neither La3+ nor Ni2+ entered the cells under resting or stimulated conditions, but both blocked Ca2+ entry. Although both Co2+ and Mn2+ entered unstimulated cells, this process was not increased by MeCh. Both Sr2+ and Ba2+ were capable of supporting a sustained increase in fura-2 fluorescence in response to MeCh, indicating that these cations can enter the cells through the agonist-regulated channels. However, Sr2+, but not Ba2+, was capable of refilling the agonist-sensitive intracellular stores. These findings demonstrate dissociation of agonist-induced Ca2+ entry from intracellular Ca2+ pool refilling and thereby provide strong support for the recently modified version of the capacitative Ca2+ entry model according to which influx into the cytoplasm occurs directly across the plasma membrane and does not require a specialized cation channel directly linking the extracellular space and the intracellular Ca2+ stores.     

Cytosolic free calcium concentrations in avian growth plate chondrocytes

Isolated avian growth plate chondrocytes convert the acetoxymethyl ester (AM) form of Fura-2 quickly and efficiently to the Ca2(+)-sensitive pentacarboxylic acid (FA) form. Control experiments indicate that the Kd for intracellular Fura-2/FA is very close to that of extracellular Fura-2/FA at the same ionic strength and pH and that the Fura-2/FA fluorescence from indicator converted by intracellular organelles is quite small. Correcting for the effects of extracellular Fura-2/FA and partial hydrolysis products has improved the accuracy of determination of intracellular [Ca2+] over earlier measurements in chondrocytes. Cytosolic [Ca2+] in isolated growth plate chondrocytes (containing cells from each maturational stage) is found to require approximately 9 hours to recover from the isolation process. After this recovery period, cytosolic [Ca2+] in these cells converges to approximately 70 nM regardless of the [Ca2+] of the recovery medium, suggesting regulation of cytosolic [Ca2+] to a set point. Chondrocytes that are separated into maturationally distinct fractions using countercurrent centrifugal elutriation show an increase in cytosolic [Ca2+] with cellular maturation. The least mature resting cells have a [Ca2+] near 57 nM, while the most mature hypertrophic cells are around 95 nM.     

Rapid mobilization of cellular Ca2+ in bovine parathyroid cells evoked by extracellular divalent cations. Evidence for a cell surface calcium receptor

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in dissociated bovine parathyroid cells using the fluorescent indicator quin-2 or fura-2. Small increases in the concentration of extracellular Ca2+ produced relatively slow, monophasic increases in [Ca2+]i in quin-2-loaded cells, but rapid and transient increases followed by lower, yet sustained (steady-state), [Ca2+]i increases in fura-2-loaded cells. The different patterns of change in [Ca2+]i reported by quin-2 and fura-2 appear to result from the greater intracellular Ca2+-buffering capacity present within quin-2-loaded cells, which tends to damp rapid and transient changes in [Ca2+]i. In fura-2-loaded parathyroid cells, other divalent cations (Mg2+, Sr2+, Ba2+) also evoked transient increases in [Ca2+]i, and their competitive interactions suggest that they all affect Ca2+ transients by acting on a common site. In contrast, divalent cations failed to cause increases in steady-state levels of cytosolic Ca2+. Low concentrations of La3+ (0.5-10 microM) depressed steady-state levels of cytosolic Ca2+ elicited by extracellular Ca2+ but were without effect on transient increases in [Ca2+]i elicited by extracellular Ca2+, Mg2+ or Sr2+, suggesting that increases in the steady-state [Ca2+]i arise from the influx of extracellular Ca2+. Mg2+- and Sr2+-induced cytosolic Ca2+ transients persisted in the absence of extracellular Ca2+ but were abolished by pretreatment with ionomycin. These results show that cytosolic Ca2+ transients arise from the mobilization of cellular Ca2+ from a nonmitochondrial pool. Extracellular divalent cations thus appear to act at some site on the surface of the cell, and this site can be considered a "Ca2+ receptor" which enables the parathyroid cell to detect small changes in the concentration of extracellular Ca2+.     

Intracellular ionic changes in normal and transformed human fibroblasts after extracellular Ca2+ deprivation.

The lowering of extracellular Ca2+ concentration in the growth medium reversibly blocks normal, but not SV40-transformed WI38 diploid fibroblasts in the early G1/G0 phase of the cell cycle. This growth response is characterized by specific changes in ionic content and transport. Ca2+ deprivation (0.03 mM) has little effect on the K+ content of either normal or transformed cells. Na+ content, however, is increased nearly 2-fold in the normal cells. This increase is presumably due to a 3-fold increase in unidirectional Na+ influx in Ca2+-deprived cells. The increased intracellular Na+ also gives rise to a nearly 3-fold enhancement of the active (ouabain-sensitive) Na+ efflux. Ca2+ deprivation causes only slight increases in Na+ influx, ouabain-sensitive Na+ efflux and intracellular Na+ in the transformed cell. In contrast, the transformed cells lose nearly 60% of their intracellular Ca2+ on deprivation, whereas normal WI38 cells lose only 10%. The data suggest that the growth arrest exhibited by the normal cell but not the transformed cell may be related to different membrane-transport and permeability changes in response to Ca2+ deprivation.     

Matrilin-4 is processed by ADAMTS-5 in late Golgi vesicles present in growth plate chondrocytes of defined differentiation state

The two aggrecanases ADAMTS-4 and ADAMTS-5 have been shown to not only play roles in the breakdown of cartilage extracellular matrix in osteoarthritis, but also mediate processing of matrilins in the secretory pathway. The matrilins are adaptor proteins with a function in connecting fibrillar and network-like components in the cartilage extracellular matrix. Cleavage resulting in processed matrilins with fewer ligand-binding subunits could make these less efficient in providing matrix cohesion. In this study, the processing and degradation of matrilin-4 during cartilage remodeling in the growth plate of the developing mouse long bones were studied in greater detail. We show that ADAMTS-5 and a matrilin-4 neoepitope, revealed upon ADAMTS cleavage, colocalize in prehypertrophic/hypertrophic chondrocytes while they are not detected in proliferating chondrocytes of the growth plate. ADAMTS-5 and the cleaved matrilin-4 are preferentially detected in vesicles derived from the Golgi apparatus. The matrilin-4 neoepitope was not observed in the growth plate of ADAMTS-5 deficient mice. We propose that in the growth plate ADAMTS-5, and not ADAMTS-4, has a physiological function in the intracellular processing of matrilins and potentially of other extracellular matrix proteins.     

Elemental analysis of growth plate cartilage by synchrotron-radiation-induced X-ray emission (SRIXE).

The elemental composition of growth plate cartilage from calf scapula has been studied by means of SRIXE. X-ray emission spectra were obtained from the resting, hypertrophic and calcified regions of cartilage; then, each element was mapped with a lateral definition of about 10 microns x 10 microns. Evidence was found for a homogeneous distribution of the elements in resting cartilage compared to changes in local concentration of some atoms in the hypertrophic-calcified tissue. In this zone Ca, Sr, Ni, Zn, S, reach the maximal concentration at the calcification front while Cu shows a uniform distribution. A Zn distribution similar to that of the Zn-containing enzyme alkaline phosphatase, the key enzyme of calcification, is found.     

Activation of contractility and occurrence of aftercontractions in Ca2+ and Sr2+-solutions during the postnatal development of the rat heart

Activation of isometric contractile force and induction of aftercontractions by different extracellular Ca- and Sr-concentrations during the early postnatal development of the rat heart were studied. In the neonatal (1-15 days) rat heart activation of contractility by [Ca]0 and [Sr]0 were similar when a dose-response curve for Ca was determined before Sr, but if the experiment was performed in the reverse order of the dose-response curve for Sr was shifted to the left. In either case the maximal developed tension was about the same. In the adult (3-5 months) rat heart [Ca]0 higher than 4 mM was inhibitory, whereas contractile force increased up to 16 mM [Sr]0 without any signs of force depression. The dose-response curve for Sr was shifted to the right compared to that of Ca and the maximal developed tension was clearly higher in Sr-solution than in Ca-solution. Aftercontractions appeared for the first time on the 13th and 24th postnatal days for 16 mM [Ca]0 and [Sr]0, respectively. In the adult rat ventricle lower Ca concentrations were needed to induce aftercontractions compared to Sr. Based upon these observations it is suggested that the appearance of aftercontractions during the third postnatal week of rat heart development is due to the maturation of intracellular Ca stores which become available for contractile activation by the development of the T-system. The absence of a negative inotropic effect in elevated Sr concentrations may be due to the slowing down of the transport processes of the sarcoplasmic reticulum (SR) by Sr and to the greater ability of the SR to store Sr over Ca.     

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice     

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice     

Transport of inorganic phosphate in primary cultures of chondrocytes isolated from the tibial growth plate of normal adolescent chickens

This report describes Pi transport activity in chondrocytes isolated from the growth plate (GP) of normal adolescent chickens grown in primary cell culture. Our recent work showed that Pi transport in matrix vesicles (MV) isolated from normal GP cartilage was not strictly Na+-dependent, whereas previously characterized Pi transport from rachitic GP cartilage MV was. This Na+-dependent Pi transporter (NaPiT), a member of the Type III Glvr-1 gene family, is expressed only transiently during early differentiation of GP cartilage, is enhanced by Pi-deficiency, and is most active at pH 6.8. Since GP mineralization requires abundant Pi and occurs under slightly alkaline conditions, it seemed unlikely that this type of Pi transporter was solely responsible for Pi uptake during normal GP development. Therefore we asked whether the lack of strict Na+-dependency in Pi transport seen in normal MV was also evident in normal GP chondrocytes. In fact, cellular Pi transport was found not to be strictly Na+-dependent, except for a brief period early in the culture. Choline could equally serve as a Na+ substitute. Activity of choline-supported Pi transport was optimum at pH 7.6-8.0. In addition, prior exposure of the cells to elevated extracellular Pi (2-3 mM) strongly enhanced subsequent Pi uptake, which appeared to depend on prior loading of the cells with mineral ions. Prevention of Pi loading by pretreatment with Pi transport inhibitors not only inhibited subsequent cellular Pi uptake, it also blocked mineral formation. Treatment with elevated extracellular Pi did not induce apoptosis in these GP chondrocytes.     

Bone growth retardation in mouse embryos expressing human collagenase 1

Cellular growth and differentiation are readouts of multiple signaling pathways from the intercellular and/or extracellular milieu. The extracellular matrix through the activation of cellular receptors transmits these signals. Therefore, extracellular matrix proteolysis could affect cell fate in a variety of biological events. However, the biological consequence of inadequate extracellular matrix degradation in vivo is not clear. We developed a mouse model expressing human collagenase (matrix metalloproteinase-1, MMP-1) under the control of Col2a1 promoter. The mice showed significant growth retardation during embryogenesis and a loss of the demarcation of zonal structure and columnar array of the cartilage. Immunological examination revealed increased degradation of type II collagen and upregulation of fibronectin and alpha(5)-integrin subunit in the transgenic cartilage. The resting zone and proliferating zone of the growth plate cartilage exhibited a simultaneous increase in bromodeoxyuridine (BrdU)-incorporated proliferating cells and terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling-positive apoptotic cells, respectively. Chondrocyte differentiation was not disturbed in the transgenic mice as evidenced by normal expression of the Ihh and type X collagen expression. These data demonstrate that type II collagen proteolysis is an important determinant for the skeletal outgrowth through modulation of chondrocyte survival and cartilagenous growth.