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PubMed1987

Warm ischemic damage to the epiphyseal growth plate: a rabbit model.

Warm ischemic growth plate damage was produced in the hindlimbs of 33 New Zealand white rabbits, who were 7 weeks old, by isolating the knee joint on a popliteal vascular pedicle. Zero, 3, 7, and 12 hours of warm ischemia were produced by pedicle occlusion. The 0- and 3-hour ischemic groups showed mild growth disturbances related to the surgical model and were not statistically different at 90 days postoperatively. The 7- and 12-hour ischemia groups showed severe growth disturbances and were statistically different from the 0- and 3-hour groups at all periods measured (p less than 0.05). Large central growth plate lesions were apparent by histologic examination. Twelve hours of ischemia produced a histologic picture identical to growth plate infarction. Increased warm ischemic time correlated with more severe growth retardation and evidence of increased damage to the central area of the growth plate histologically.

Animals↗

PubMed2006

Expression of bone morphogenic proteins and receptors at the injured growth plate cartilage in young rats.

The injured growth plate cartilage is often repaired by bony tissue, resulting in impaired bone growth in children. Bone morphogenic proteins (BMPs) are important for bone fracture repair, and as a step to characterize potential involvement of BMPs in bony repair of injured growth plate, expression of BMPs and receptors (BMP-R) was examined by quantitative RT-PCR and immunohistochemistry in rat injured tibial growth plate. During the inflammatory response on day 1, slightly increased expression of BMP-3, BMP-4, BMP-R1a, and BMP-R2 was observed, with immunostaining seen among inflammatory cells at the injury site. During mesenchymal infiltration and osteogenic responses on days 3-14, moderately increased expression of BMP-2, -3, -4, -7, and BMP-R1a was found, with immunostaining observed among infiltrated mesenchymal cells and differentiated osteoblasts lining bony trabeculae. During maturation phase on days 14-25, only BMP-7 was seen upregulated slightly and was localized in osteoblasts and marrow cells at the injury site. The temporospatial expression of BMPs and receptors at the injured growth plate suggests potential involvement of BMP-3 and -4 in regulating the inflammatory response or as its mediators in modulating downstream events, and BMP-2, -3, -4, and -7 in the fibrogenic and osteogenic responses, and BMP-7 in bone remodeling at the injured growth plate.

Age Factors↗

PubMed1989

Condensation of hypertrophic chondrocytes at the chondro-osseous junction of growth plate cartilage in Yucatan swine: relationship to long bone growth.

Chondrocytes of the cartilaginous growth plate are found in a spatial gradient of cellular differentiation beginning with cellular proliferation and ending with cellular hypertrophy. Although it is recognized that both proliferation and hypertrophy contribute significantly to overall bone growth, mechanisms acting on the chondrocyte to control the timing, the rate, and the extent of hypertrophy are poorly understood. Similarly, mechanisms acting on the terminal chondrocyte to cause its death at the chondro-osseous junction have not been investigated. In this study we examine the condensation of terminal hypertrophic chondrocytes in proximal and distal radial growth plates of Yucatan swine at 4 weeks of age. The animals were raised in a controlled environment where activity and feeding patterns were synchronized to a given time in the light/dark cycle. We analyzed cellular condensation both as a function of circadian rhythms in a 24-hr time period, and as a function of overall rate of growth. The data suggest that the magnitude of circadian influences on long bone growth is significantly damped at the level of the hypertrophic chondrocyte compared to that seen by previous investigators studying circadian influences on chondrocytic proliferation. Secondly, the condensation of hypertrophic chondrocytes at the chondro-osseous junction varies inversely with rate of growth in length of the bone. At any time period, a higher percentage of terminal chondrocytes in the condensed form was found in the slower-growing of the two growth plates. We relate these findings to current hypotheses concerning controls of chondrocytic hypertrophy and possible controls over the timing of hypertrophic cell death.

Animals↗

PubMed1996

Differential growth by growth plates as a function of multiple parameters of chondrocytic kinetics.

Differential elongation of growth plates is the process by which growth-plate chondrocytes translate the same sequence of gene regulation into the appropriate timing pattern for a given rate of elongation. While some of the parameters associated with differential growth are known, the purpose of this study was to test the hypothesis that eight independent variables are involved. We tested this hypothesis by considering four different growth plates in 28-day-old Long-Evans rats. Temporal parameters were provided by means of oxytetracycline and bromodeoxyuridine labeling techniques. Stereological parameters were measured with standard techniques. For all four growth plates, the calculated number of new chondrocytes produced per day approximated the number of chondrocytes lost per day at the chondro-osseous junction. This suggests that the proposed equations and associated variables represent a comprehensive set of variables defining differential growth. In absolute numbers, the proximal tibial growth plate produced about four times as many chondrocytes per day as the proximal radial growth plate (16,400 compared with 3,700). In the proximal tibia, 9% of growth is contributed by cellular division; 32%, by matrix synthesis throughout the growth plate; and 59%, by chondrocytic enlargement during hypertrophy. In the more slowly elongating growth plates, the relative contribution to elongation from cellular enlargement decreases from 59 to 44%, with a relative increase in contribution from matrix synthesis ranging from 32% in the proximal tibia 49% in the proximal radius. This study suggests that differential growth is best depicted as a complex interplay among cellular division, matrix synthesis, and cellular enlargement during hypertrophy. Differential growth is best explained by considering a set of eight independent variables, seven of which vary from growth plate to growth plate. Thus, this study confirms the importance of cellular hypertrophy during elongation and adds to our understanding of the importance of locally mediated regulatory systems controlling growth-plate activity.

Animals↗

PubMed1985

Calcitonin stimulates maturation of mammalian growth plate cartilage.

To determine whether calcitonin (CT) might effect maturation of mammalian growth plate cartilage, we administered salmon CT (sCT) to young rats and used the growth plate from the distal metatarsal as our in vivo growth plate model. Growth plate alkaline phosphatase activity and histological examination were assessed after 3 days of sCT treatment. Alkaline phosphatase activity, a marker of hypertrophied chondrocytes, increased 85%, and the zone of maturation enlarged in rats receiving sCT. In addition, sCT treatment was associated with an increased growth plate 35SO4 incorporation 84% above animals receiving buffer alone. We tested whether this might be a direct effect of CT by using an in vitro model, the growth plate from the fetal pig scapula. Organ culture of these cartilages in serum-free medium with and without sCT (1 U/ml) was performed for 3 days. sCT stimulated alkaline phosphatase activity 49% above growth plates incubated in medium alone. Furthermore, sCT treatment increased the number of hypertrophied chondrocytes leading to widening of the zone of maturation. These studies suggest that CT might have a role growing mammals by promoting maturation of growth plate cartilage.

Alkaline Phosphatase↗

PubMed2005

Localization of matrix metalloproteinases, (MMPs) their tissue inhibitors, and vascular endothelial growth factor (VEGF) in growth plates of children and adolescents indicates a role for MMPs in human postnatal growth and skeletal maturation.

Numerous studies have focused on the expression, regulation, and biological significance of matrix metalloproteinases (MMPs) in the growth plate. Findings in mouse knockout models and in vitro data from various species indicate that MMPs not only degrade extracellular matrix components but may regulate the activity of local growth factors. In this study we investigated the presence, distribution, and activity of various MMPs and inhibitors, tissue transglutaminase (tTG or TG2) and vascular endothelial growth factor (VEGF) in the human child and adolescent growth plates by means of immunohistochemistry and gelatin zymography. Tissue was derived during orthopedic surgery (epiphysiodesis) in two prepubertal and four pubertal patients.MMP-2 and MMP-14 were present in reserve cell chondrocytes. MMP-14 was the most prominent MMP within all zones of the growth plate including proliferating chondrocytes. MMP-1 and MMP-13 (collagenases 1 and 3), MMP-9 (gelatinases B), MMP-10, and MMP-11 (stromelysins) and VEGF were positive in hypertrophic chondrocytes and osteoblasts. MMP-2 showed the same expression pattern but was negative in osteoblasts. Osteoclasts stained positive for MMP-9, MMP-2, and TG2. Tissue inhibitor of MMP (TIMP)-1 was present in all zones of the growth plate, osteoblasts, and osteoclasts; TIMP-2 was found in hypertrophic chondrocytes and osteoblasts. In summary, the presence of MMPs, TIMPs, TG2, and VEGF in our study indicated that the MMPs are relevant in growth plate physiology during the postnatal period in humans. The specific location of MMP expression within the growth plate may be the basis for further studies on the role of MMPs in the local regulation of chondrocyte differentiation, proliferation, and ossification at the chondroosseus junction.

Adolescent↗

PubMed2004

Chondromodulin-I expression in the growth plate of young uremic rats.

BACKGROUND: Growth retardation of chronic renal failure is associated with alterations in the growth plate suggestive of a disturbed chondrocyte maturation process and abnormal vascular invasion at the chondro-osseous interphase. Chondromodulin I (ChM-I) is a potent cartilage-specific angiostatic factor. Its pattern of expression in the uremic rat growth plate is unknown. Persistence of ChM-I synthesis and/or imbalance between ChM-I and vascular endothelial growth factor (VEGF) expressions might play a role in the alterations of uremic growth plate. METHODS: Growth cartilage ChM-I expression was investigated by immunohistochemistry, in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) in growth-retarded young uremic rats (UREM), control rats, fed ad libitum (SAL) or pair-fed with the UREM group (SPF), and uremic rats treated with growth hormone (UREM-GH). VEGF expression was analyzed by immunohistochemistry. RESULTS: ChM-I and ChM-I mRNA were confined to the proliferative and early hypertrophic zones of growth cartilage. A similar number of chondrocytes per column was positive for ChM-I in the 4 groups. In accordance with the elongation of the hypertrophic stratum in uremia, the distance (X+/-SEM, microm) between the extracellular ChM-I signal and the metaphyseal end of growth cartilage was higher (P < 0.003) in UREM (236 +/- 40) and UREM-GH (297 +/- 17) than in SAL (92 +/- 7) and SPF (113 +/- 6). No differences in ChM-I expression were appreciated by RT-PCR. Similar VEGF positivity was observed in the hypertrophic chondrocytes of all groups. CONCLUSION: In experimental uremia, expansion of growth cartilage does not result from increased or persistent expression of ChM-I or from reduced VEGF expression at the cartilage-metaphyseal bone interphase. GH treatment does not modify ChM-I and VEGF expressions.

Animal Nutritional Physiological Phenomena↗

PubMed1994

Characterisation of articular and growth plate cartilage collagens in porcine osteochondrosis.

The articular and growth plate cartilages of osteochondrotic pigs were examined and compared with those from clinically normal animals. Both types of osteochondrotic cartilage showed considerable localised thickening apparently due to a lack of ossification. Histological examination of cartilage lesions demonstrated a breakdown in the normal pattern of chondrocyte maturation. Articular cartilage lesions lacked mature clones of chondrocytes in the calcifying region. Growth plate cartilage showed an accumulation of disorganised hypertrophic chondrocytes rather than the well-defined columns seen in normal tissue. The overall percentages of collagen in osteochondrotic lesions from both articular and growth plate cartilage were significantly reduced compared with levels in unaffected cartilage. There were substantial increases in the proportion of type I collagen in lesions from both osteochondrotic articular and growth plate cartilages and a reduction in the proportion of type II collagen. Type X collagen was detected in osteochondrotic but not normal articular cartilage. The proportion of type X collagen was unchanged in osteochondrotic growth plate cartilage. The levels of the collagen cross-links, hydroxylysylpyridinoline, hydroxylysyl-ketonorleucine and dehydrohydroxylysinonorleucine were radically reduced in samples from osteochondrotic growth-plate cartilage lesions when compared with normal tissue. Less dramatic changes were observed in articular cartilage although there was a significant decrease in the level of hydroxylysylketonorleucine in osteochondrotic lesions. Immunofluorescence examination of osteochondrotic lesions showed a considerable disruption of the organisation of the collagenous components within both articular and growth-plate cartilages. Normal patterns of staining of types I and VI collagen seen at the articular surface in unaffected tissue were replaced by a disorganised, uneven stain in osteochondrotic articular cartilage lesions. Incomplete removal of cartilage at the ossification front of osteochondrotic growth plate was demonstrated by immunofluorescence staining of type IX collagen. Type X collagen was produced in the matrix of the calcifying region of osteochondrotic articular cartilage by small groups of hypertrophic chondrocytes, but was not detected in normal articular cartilage. The distribution of type X collagen was unchanged in osteochondrotic growth plate cartilage.

Animals↗

PubMed1986

Histopathology of growth plate changes in induced abnormal bone growth in lambs.

Unilateral hip excision arthroplasty in lambs resulted in osteochondrosis developing in distal tibiae and metatarsi of the contralateral pelvic limb. The physeal cartilage of distal tibiae and metatarsi developed clefts which probably represent shear failure. The histopathology of the meataphyseal defects differed between the medial and lateral growth plate sectors. Medially, the lesions were typical of physeal osteochondrosis in other mammals, with disorganized chondrocyte columns, cell death and matrix necrosis. Laterally, metaphyseal defects were predominantly fibrous, and islands of bone formed in physeal cartilage from re-established endochondral ossification. Both medial and lateral metaphyseal defects were associated with disturbed primary spongiosa formation. It is suggested that, in some instances at least, traumatic physeal clefts, resulting from shear failure in previously normal growth cartilage, may interfere with endochondral ossification and produce osteochondrosis.

Animals↗

PubMed1994

Basic fibroblast growth factor binds to heparan sulfate in the extracellular matrix of rat growth plate chondrocytes.

Recent studies have demonstrated that basic fibroblast growth factor (bFGF) plays a key role in the terminal differentiation of growth plate chondrocytes during endochondral ossification. We therefore examined the binding of [125I]bFGF to an extract of extracellular matrix (ECM) of rat growth plate. Using a solid phase binding assay, binding of [125I]bFGF to ECM was demonstrated to be specific and saturable at 0.5-1 microgram/ml of bFGF. Scatchard analysis demonstrated the presence of a single class of binding sites with an apparent Kd of 14 nM. The binding of [125I]bFGF to rat growth plate ECM was inhibited by the addition of heparin, heparan sulfate, and dermatan sulfate. The binding was reversible as these glycosaminoglycans were also effective at displacement of bound [125I]bFGF from rat growth plate ECM. Chondroitin 4- or 6-sulfate had no effect on either binding or displacement when added at the same concentrations. Preincubation of the rat growth plate ECM with heparinase or heparitinase resulted in a reduction of the binding of [125I]bFGF to the ECM. Furthermore, these enzymes were able to significantly displace bound growth factor. In contrast, chondroitinase ABC or AC failed to displace bound [125I]bFGF from the ECM. Similar results were obtained when matrix derived from rat growth plate tissue was used for the binding studies. The results demonstrate that bFGF binds to a heparan sulfate in matrix produced by rat growth plate chondrocytes and matrix extracted from rat growth plate and suggest that this glycosaminoglycan may serve as a storage depot for this growth factor during endochondral ossification.

Alkaline Phosphatase↗

PubMed1998

Local disappearance of epiphyseal growth plates in rats with hypervitaminosis A.

Epiphyseal growth plates of proximal tibiae in rats with high doses of vitamin A (V-A) were observed. Of 4 groups, each consisting of 5 rats, three groups were given V-A at doses, IU/100 g by body weight/day, of 50,000, 100,000, and 150,000, respectively. The other group rats were given no V-A (control). Rats were administered V-A for the 5 days from 4 weeks after birth and sacrificed at 12 weeks after birth. Three rats of the 150,000 IU group died during the period of observation. The decalcified sections were stained with hematoxylin-eosin or toluidine blue. In the ground sections, microradiography, backscattered electron imaging, and energy-dispersive X-ray microanalysis were performed. These observations suggest that the local disappearance of epiphyseal growth plates under high doses of V-A goes in the order of the increased doses through the process of (1) calcified cartilage areae appearing in the resting cell zone, (2) some of the calcified areae extending in the growth plate towards the diaphysial side, (3) bone tissue replacing the calcified areae, and (4) the local disappearing of the growth plate. Such a local disappearance may be formed in the stressed proximal regions of tibiae.

Animals↗

PubMed2001

The chondrogenic transcription factor Sox9 is a target of signaling by the parathyroid hormone-related peptide in the growth plate of endochondral bones.

In the growth plate of endochondral bones, parathyroid hormone (PTH)-related peptide (PTHrP) regulates the rate of chondrocyte maturation from prehypertrophic chondrocytes to hypertrophic chondrocytes. Using an antibody specific for Sox9 phosphorylated at serine 181 (S(181)), one of the two consensus protein kinase A phosphorylation sites of Sox9, we showed that the addition of PTHrP strongly increased the phosphorylation of SOX9 in COS7 cells transfected with both SOX9- and PTH/PTHrP receptor-expressing vectors. PTHrP also increased the SOX9-dependent activity of chondrocyte-specific enhancers in the gene for type II collagen (Col2a1) in transient transfection experiments. This increased enhancer activity did not occur with a Sox9 mutant harboring serine-to-alanine substitutions in its two consensus protein kinase A phosphorylation sites. Consistent with these results, PTHrP also increased Col2a1 mRNA levels in rat chondrosarcoma cells as well as 10T1/2 mesenchymal cells transfected with a PTH/PTHrP receptor expressing plasmid. No phosphorylation of Sox9 at S(181) was detected in prehypertrophic chondrocytes of the growth plate or any chondrocytes of PTH/PTHrP receptor null mutants. In contrast in wild-type mouse embryos, previous immunohistochemistry experiments indicated that Sox9 phosphorylated at S(181) was detected almost exclusively in chondrocytes of the prehypertrophic zone. Sox9, regardless of the phosphorylation state, was present in all chondrocytes of both genotypes except hypertrophic chondrocytes. Our results indicated that Sox9 is a target of PTHrP signaling in prehypertrophic chondrocytes in the growth plate. We hypothesize that Sox9 mediates at least some effects of PTHrP in the growth plate and that the PTHrP-dependent increased transcriptional activity of Sox9 helps maintain the chondrocyte phenotype of cells in the prehypertrophic zone and inhibits their maturation to hypertrophic chondrocytes.

Active Transport, Cell Nucleus↗

PubMed1989

Direct administration of testosterone increases rat tibial epiphyseal growth plate width.

Local injection of hormones into the tibial epiphyseal growth plate offers a possible model to answer whether sex steroids can affect bone growth directly. To answer this question, we injected different doses of testosterone enanthate (4, 40, 120 and 400 micrograms/100 g of rat weight) once into the tibial epiphyseal growth plate of castrated 35-day-old male rats. The contralateral tibia was injected with sesame oil and served as control. All animals were sacrificed at age 42 days. Tibias were removed for measurement of epiphyseal growth plate width and blood was collected for measurement of serum IGF-I and testosterone. The lower doses of testosterone enanthate (4, 40 and 120 micrograms/100 g) did not produce any significant change in epiphyseal growth plate width. Testosterone at the largest dose tested (400 micrograms/100 g) increased epiphyseal growth plate width by about 15% compared to control (p less than 0.01). At this dose, serum testosterone was not increased, suggesting that the effect on epiphyseal growth plate width was not due to higher systemic testosterone concentrations. No differences in IGF-I levels were observed among the groups. We conclude that direct administration of testosterone enanthate at a dose of 400 micrograms/100 g into the rat tibial epiphyseal growth plate can increase epiphyseal growth plate width.

Animals↗

PubMed1994

Expression of collagens I, II, X, and XI and aggrecan mRNAs by bovine growth plate chondrocytes in situ.

The cells responsible for skeletal growth are the chondrocytes of the cartilaginous growth plate. These cells differentiate through a series of maturational stages, establishing different zones in the growth plate. Among the major functions of these cells is the production of appropriate extracellular matrix, primarily composed of collagens and proteoglycans. To determine whether matrix synthesis varies with respect to maturational stage and in which cell populations different collagens are expressed, bovine growth plates were analyzed by in situ hybridization to mRNA and by Northern blot hybridization. The most abundant collagen mRNA in the growth plate was type-II collagen. This mRNA was present at relatively low levels in the most immature cells of the growth plate but increased several-fold as cells entered the proliferative stage and remained high through subsequent phases of maturation. Type-XI collagen mRNA and mRNA for the cartilage-characteristic proteoglycan, aggrecan, were codistributed with the type-II collagen mRNA; however, both were present in much smaller quantities. Type-X procollagen mRNA was localized to chondrocytes late in their maturation and was expressed at levels similar to the expression of type-II collagen. In situ hybridization of serial sections revealed that growth plate chondrocytes in their more mature stages contain both type-II and type-X collagen mRNA. Type-I collagen mRNA was not observed in growth plate chondrocytes at any maturational stage; rather, it was localized to a morphologically distinct population of cells attached to calcifying cartilage septa in the region of vascular invasion.(ABSTRACT TRUNCATED AT 250 WORDS)

Aggrecans↗

PubMed2005

Thyroid hormone regulates heparan sulfate proteoglycan expression in the growth plate.

Thyroid hormone is essential for normal skeletal development. Hypothyroidism is associated with growth arrest, failure of chondrocyte differentiation, and abnormal matrix synthesis. Thyroid hormone modulates the Indian hedgehog/PTHrP feedback loop and regulates fibroblast growth factor (FGF)/FGF receptor signaling. Because heparan sulfate (HS) proteoglycans (Prgs) (HSPGs) are absolutely required by these signaling pathways, we have investigated whether thyroid status affects HSPG expression within the growth plate. Tibial growth plate sections were obtained from 12-wk-old rats rendered euthyroid, thyrotoxic, or hypothyroid at 6 wk of age, 14-d-old congenitally hypothyroid Pax8-null mice, and TRalpha/TRbeta double-null mice lacking all thyroid hormone receptors. HS and chondroitin sulfate Prg expression was determined by immunohistochemistry using three monoclonal antibodies. There was increased HS staining in growth plates from hypothyroid animals predominantly within the extracellular matrix of reserve and proliferative zones. Cellular HS staining was also increased particularly in prehypertrophic chondrocytes. T3 regulation of HSPG core protein and HS synthetic and modification enzyme expression was studied in ATDC5 cells using semiquantitative RT-PCR. Thyroid hormone negatively regulated expression of the core protein Gpc6, the polymerase Ext1, and the modification enzyme Hs6st2. These studies demonstrate that the expression and distribution of growth plate Prgs are regulated by thyroid hormone, and the regulation of HSPG expression provides an important additional link between FGF and Indian hedgehog signaling and T3. These novel observations suggest that the cartilage matrix and especially HSPGs are critical mediators of the skeletal response to thyroid hormone.

Animals↗

PubMed2000

Suppression of growth plate chondrocyte proliferation by corticosteroids.

Growth depression is a side effect of high-dose glucocorticoid therapy in childhood. It is partially mediated by alterations of the somatotropic hormone axis and partially by direct local effects on growth plate chondrocytes. The mechanisms of interaction of corticosteroids and somatotropic and calciotropic hormones at the cellular level were recently investigated in more detail, using experimental models of primary cultures of growth plate chondrocytes. In proliferative chondrocytes, growth hormone (GH) and the calciotropic hormones parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D [1alpha,25(OH)2D3] increase cell proliferation via stimulation of paracrine insulin-like growth factor-I (IGF-I) secretion. Corticosteroids decreased GH, and PTH or 1alpha,25(OH)2D3 stimulated cell growth in a dose-dependent manner. Corticosteroids in high doses reduced the expression of the GH receptor and type 1 IGF receptor. But the main antiproliferative molecular effect of corticosteroid was the reduction in basal and hormone-stimulated IGF-I secretion. The in vitro results are in accordance with the observation in animal experiments and in children treated with corticosteroids, demonstrating that the growth-depressing effect of corticosteroids can be compensated for by supraphysiological doses of GH or IGF-I.

Adrenal Cortex Hormones↗

PubMed2001

Stathmin levels in growth plate chondrocytes are modulated by vitamin D3 metabolites and transforming growth factor-beta1 and are associated with proliferation.

Stathmin is a highly conserved, phosphorylated cytosolic protein that is found at decreased levels in all cells as they become more terminally differentiated, or when they decrease in their rate of proliferation. This study examined the hypothesis that stathmin levels in growth plate chondrocytes decreases as endochondral maturation increases. To test this hypothesis, we used a costochondral growth plate chondrocyte cell culture model. Cells derived from the resting zone (RC) express twice as much stathmin mRNA in culture and have twice as much stathmin protein as cells derived from the post proliferative growth zone ([GC]; prehypertrophic and upper hypertrophic cell zones). Stathmin levels in vivo were assessed by immunohistochemistry. To assess the effects of agents that modulate proliferation and differentiation, RC and GC chondrocytes were cultured in the presence of 10(-10) to 10(-8) M 1alpha,25-(OH)2D3, which regulates proliferation in both cell types but affects differentiation of only GC cells, or 10(-9) to 10(-7) M 24R,25-(OH)2D3, which regulates differentiation and maturation of RC cells but decreases proliferation of GC cells. In addition, RC cells were treated with 0.44 or 0.88 ng/mL of recombinant human transforming growth factor beta1 (rhTGF-beta1), which stimulates proliferation of RC cells and regulates proteoglycan production, but not alkaline phosphatase activity. Stathmin protein levels were determined using quantitative immunoblots, with recombinant human stathmin as a standard. The results show that stathmin levels are associated with proliferation. Proliferating chondrocytes in vivo exhibited higher levels of immunoreactive stathmin than either RC or GC cells in the growth plate. In culture, 1alpha,25-(OH)2D3 caused a dose-dependent decrease in stathmin in RC and GC cells within 24 h. 24R, 25-(OH)2D3 also reduced stathmin levels in GC cells within 24 h but only affected RC cells after prolonged exposures (96 h), at which time RC cells express a GC-like phenotype. rhTGF-beta1 caused an increase in stathmin levels in RC cells. Stathmin levels are sensitive to protein kinase C (PKC) in other cells. Inhibition of PKC with chelerythrine had no effect on the response of RC cells to 1alpha,25-(OH)2D3 but it blocked the effect of rhTGF-beta1, indicating that decreases in stathmin by vitamin D3 metabolites may not be modulated by PKC, whereas increases in stathmin via rhTGF-beta1 may be regulated via a PKC-dependent mechanism. These results support the hypothesis that constitutively expressed levels of stathmin are related to cell maturation state and that they are modulated by factors that regulate proliferation.

24,25-Dihydroxyvitamin D 3↗

PubMed2004

Growth factor regulation of human growth plate chondrocyte proliferation in vitro.

Linear growth occurs as the result of growth plate chondrocytes undergoing proliferative and hypertrophic phases. Paracrine feedback loops that regulate the entry of chondrocytes into the hypertrophic phase have been shown and similar pathways likely exist for the proliferative phase. Human long-bone growth plate chondrocytes were cultured in vitro. The proliferative effects of a variety of factors were determined by [3H]thymidine uptake and the gene expression profile of these cells was determined by DNA microarray analysis. Serum, insulin-like growth factor (IGF)-I and -II, transforming growth factor-beta (TGF-beta, fibroblast growth factor (FGF)-1, -2, and -18, and platelet-derived growth factor (PDGF)-BB were potent stimulators of proliferation. FGF-10, testosterone, and bone morphogenetic proteins (BMP)-2, -4, and -6 inhibited proliferation. Microarray analysis showed that the genes for multiple members of the IGF-I, TGF-beta, FGF, and BMP pathways were expressed, suggesting the presence of autocrine/paracrine pathways that regulate the proliferative phase of growth plate-mediated growth.

Adolescent↗