Thursday, March 7, 2013

The Resting Zone of the Growth Plate

Within the resting zone of the growth plate are stem-like cells which means they are like stem cells but only have limited proliferative capacity.  If we can characterize these resting zone cells we can know more about how to initiate the first stage of the growth plate.

Identification of target genes for wild type and truncated HMGA2 in mesenchymal stem-like cells.

"The HMGA2{up in LSJL} gene [codes] for an architectural transcription factor involved in mesenchymal embryogenesis.
We have over-expressed wild type and truncated HMGA2 protein in an immortalized mesenchymal stem-like cell (MSC) line, and investigated the localisation of these proteins and their effects on differentiation and gene expression patterns.
Over-expression of both transgenes blocked adipogenic differentiation of these cells, and microarray analysis revealed clear changes in gene expression patterns, more pronounced for the truncated protein. Most of the genes that showed altered expression in the HMGA2-overexpressing cells fell into the group of NF-kappaB-target genes, suggesting a central role for HMGA2 in this pathway. Of particular interest was the pronounced up-regulation of SSX1, already implicated in mesenchymal oncogenesis and stem cell functions, only in cells expressing the truncated protein. Furthermore, over-expression of both HMGA2 forms was associated with a strong repression of the epithelial marker CD24, consistent with the reported low level of CD24 in cancer stem cells.:
We conclude that the c-terminal part of HMGA2 has important functions at least in mesenchymal cells, and the changes in gene expression resulting from overexpressing a protein lacking this domain may add to the malignant potential of sarcomas."

"There were several genes up-regulated by HMGA2WT and down- regulated in cells expressing the truncated form, such as FGF13, EHF, HCLS1, MEST, G0S2 and PTPRN2."<-Since the truncated form of HMGA2 can increase height these genes may be important.

Genes downregulated in HMGA2WT-transgenic also downregulated in LSJL:


Genes downregulated in HMGA2Ttruncated also downregulated in LSJL:


"over-expression of truncated HMGA2 induces a more mesenchymal (stem-like) phenotype, characterized by resistance toward differentiation, over-expression of SSX1, lost expression of certain epithelial markers and strengthened expression of mesenchymal markers."

Differential expression of phenotype by resting zone and growth region costochondral chondrocytes in vitro.

"Chondrocytes derived from the resting cell zone and adjacent growth zone of rat costochondral cartilage were compared for retention of phenotype in culture. At third passage confluence, two cell populations differ morphologically and biochemically. Resting zone cells are fibroblast-like, with smooth cell membranes and little rough endoplasmic reticulum. Growth zone cells are more polygonal, smaller in diameter, with numerous cytoplasmic extensions of the plasma membranes and abundant rough endoplasmic reticulum. Both cell populations produce matrix vesicles that are comparable morphologically to matrix vesicles isolated enzymatically from epiphyseal cartilage. While membrane vesicles are released into the media by cells derived from the resting zone as well as from the growth cartilage, alkaline phosphatase activity is enriched in media vesicles produced by growth cartilage cells. Alkaline phosphatase enriched vesicles appear to be preferentially incorporated into the extracellular matrix. Both the plasma membrane marker enzyme activity and the membrane phospholipid composition are differentially expressed in matrix vesicles and plasma membranes and are cell specific. Matrix vesicles produced by resting zone cells are enriched in alkaline phosphatase, 5'-nucleotidase, ouabain sensitive Na+/K+ ATPase and cardiolipin when compared to the cell membrane. In addition, the plasma membranes of these cells contain more phosphatidylcholine plus sphingomyelin than do growth cartilage plasma membranes. Resting zone cell matrix vesicles have less phosphatidylethanolamine than do vesicles from growth cartilage cultures. Matrix vesicles produced by growth cartilage cells contain one proteolipid at 43,000 Mr which comigrates with plasma membrane proteolipid and an additional proteolipid at approximately 3,000 Mr. These data indicate that both cells retain differential expression of phenotype in culture and that one expression of this phenotype is production of specific extracellular matrix vesicles."

"Growth cartilage chondrocyte plasma membranes exhibit higher 5'-nucleotidase activity than do resting cell membranes"

"The resting zone cells membranes contain more phosphatidylcholinc plus sphingomyelin than do the growth zone chondrocyte membranes"

Transforming growth factor-beta1 regulation of resting zone chondrocytes is mediated by two separate but interacting pathways.

" transforming growth factor-beta1 (TGF-beta1) stimulates protein kinase C (PKC) via a mechanism that is independent of phospholipase C or tyrosine kinase, but involves a pertussis toxin-sensitive G-protein. Maximal activation occurs at 12 h and requires new gene expression. To understand the signaling pathways involved, resting zone chondrocytes were incubated with TGF-beta1 and PKC activity was inhibited with chelerythrine, staurosporine or H-7. [(35)S]Sulfate incorporation was inhibited, indicating that PKC mediates the effects of TGF-beta1 on matrix production. However, there was little, if any, effect on TGF-beta1-dependent increases in [(3)H]thymidine incorporation, and TGF-beta1-stimulated alkaline phosphatase was unaffected, indicating that these responses to the growth factor are not regulated via PKC. TGF-beta1 caused a dose-dependent increase in prostaglandin E(2) (PGE(2)) production which was further increased by PKC inhibition. The increase was regulated by TGF-beta1-dependent effects on phospholipase A(2) (PLA(2)). Activation of PLA(2) inhibited TGF-beta1 effects on PKC, and inhibition of PLA(2) activated TGF-beta1-dependent PKC. Exogenous arachidonic acid also inhibited TGF-beta1-dependent increases in PKC. The effects of TGF-beta1 on PKC involve genomic mechanisms, but not regulation of existing membrane-associated enzyme, since no direct effect of the growth factor on plasma membrane or matrix vesicle PKC was observed.  TGF-beta1 modulates its effects on matrix production through PKC, but its effects on alkaline phosphatase are mediated by production of PGE(2) and protein kinase A (PKA). Inhibition of PKA also decreases TGF-beta1-dependent proliferation. We have previously shown that PGE(2) stimulates alkaline phosphatase through its EP2 receptor, whereas EP1 signaling causes a decrease in PKC. Thus, there is cross-talk between the two pathways."

"Resting zone chondrocytes synthesize TGF-β1 in latent form and store it in their extracellular matrix as a 290 kDa complex consisting of latent TGF-β1, latent TGF-β1 binding protein-1 and the latency-associated peptide. Extracellular matrix vesicles produced by these cells can activate latent TGF-β1 when they are exposed to 1,25-(OH)2D3. The interrelationship of TGF-β1 action and vitamin D metabolites is also demonstrated by the fact that TGF-β1 causes resting zone cells to produce increased 1,25-(OH)2D3 within 1 h and increased 24,25-(OH)2D3 at 24 h, which is correlated with TGF-β1-dependent downregulation of the 1α-hydroxylase and upregulation of the 24-hydroxylase in these cells"

"PGE2 has multiple effects on the chondrocytes, promoting differentiation and anabolic responses via cAMP production and PKC activity"

" Resting zone cells have both EP1 and EP2 receptors, as well as an EP1 variant, EP1v. The increase in cAMP leads to increased PKA activity. The importance of this pathway in the response to TGF-β1 is evident in the decrease in proliferation following treatment of the cells with TGF-β1 and the PKA inhibitor, H-8."

Direct effects of 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 on growth zone and resting zone chondrocyte membrane alkaline phosphatase and phospholipase-A2 specific activities.

"1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 differentially affect the specific activity of alkaline phosphatase (ALPase) and phospholipase-A2 (PLA2) of plasma membranes and extracellular matrix vesicles produced by costochondral reserve zone and growth zone cartilage chondrocytes in culture. In the present study, growth zone and cartilage and reserve zone matrix vesicles and plasma membranes were isolated from confluent chondrocyte cultures and incubated with hormone for 3 and 24 h in vitro. Addition of 1,25-(OH)2D3 to GC matrix vesicles and plasma membranes resulted in dose-dependent increases in ALPase and PLA2 specific activities in both membrane fractions. Addition of 24,25-(OH)2D3 to RC membrane fractions stimulated matrix vesicle ALPase at 10(-7) and 10(-8) M and plasma membrane ALPase at 10(-8) M only. However, 24,25-(OH)2D3 inhibited matrix vesicle and plasma membrane PLA2 activity. The effects of the vitamin D metabolites were noticed after both 3 and 24 h. Neither hormone metabolite had any effect on these enzymes in membrane fractions from cultures of neonatal rat muscle mesenchymal cells, which do not calcify their matrix in vivo. 1,25-(OH)2D3 and 24,25-(OH)2D3 can directly affect chondrocyte membrane enzymes without genomic influence or protein synthesis and that membrane response depends on the stage of chondrocyte differentiation. Changes in PLA2 activity may change membrane fluidity and may be a mechanism by which the hormones affect cell membranes."

"Enzymes present in membranes isolated from the less differentiated mesenchymal cells do not respond to either vitamin D3 metabolite tested, although both metabolites stimulate ALPase gene expression in cultures of these cells"

Treatment of resting zone chondrocytes with bone morphogenetic protein-2 induces maturation into a phenotype characteristic of growth zone chondrocytes by downregulating responsiveness to 24,25(OH)2D3 and upregulating responsiveness to 1,25-(OH)2D3.

"To determine if bone morphogenetic protein-2 (BMP-2) can induce the endochondral maturation of resting zone (RC) chondrocytes, confluent fourth-passage cultures of these cells were pretreated for 24, 36, 48, 72, or 120 h with recombinant human BMP-2. At the end of pretreatment, the media were replaced with new media containing 10(-10)-10(-8) M 1,25-(OH)2D3 or 10(-9)-10(-7) M 24,25-(OH2)D3 and the cells incubated for an additional 24 h. This second treatment was chosen, because prior studies had shown that the more mature growth zone (GC) chondrocytes and RC cells respond to 1,25-(OH)2D3 and 24,25-(OH)2D3 in distinctly different ways with respect to the parameters examined. The effect of BMP-2 pretreatment on cell maturation was assessed by measuring alkaline phosphatase specific activity (ALPase). In addition, changes in matrix protein production were assessed by measuring collagen synthesis, as well as [35S]-sulfate incorporation into proteoglycans. When RC cells were pretreated for 72 or 120 h with BMP-2, treatment with 1,25-(OH)2D3 caused a dose-dependent increase in ALPase specific activity and collagen synthesis, with no effect on proteoglycan sulfation. RC cells pretreated with 1,25-(OH)2D3 responded like RC cells that had not received any pretreatment. RC cells normally respond to 24,25-(OH)2D3; however, RC cultures pretreated for 72 or 120 h with BMP-2 lost their responsiveness to 24,25-(OH)2D3. These results indicate that BMP-2 directly regulates the differentiation and maturation of RC chondrocytes into GC chondrocytes. These observations support the hypothesis that BMP-2 plays a significant role in regulating chondrocyte maturation during endochondral ossification."

"Resting zone cells exhibit greater sensitivity to BMP-2 than do cells derived from the prehypertrophic and upper hypertrophic zones"

Treatment of resting zone chondrocytes with 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] induces differentiation into a 1,25-(OH)2D3-responsive phenotype characteristic of growth zone chondrocytes.

"rat costochondral cartilage chondrocytes isolated from the growth zone (GC) respond to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], whereas those from the resting zone (RC) respond to 24,25-(OH)2D3[the inactive form of Vitamin D]. The aim of the present study was to determine whether 24,25-(OH)2D3 induces differentiation of RC cells into a 1,25-(OH)2D3-responsive GC phenotype. To do this, confluent, fourth passage RC chondrocytes were pretreated for 24, 36, 48, 72, and 120 h with 10(-7) M 24,25-(OH)2D3. The medium was then replaced with new medium containing 10(-10) to 10(-8) M 1,25-(OH)2D3, and the cells were incubated for an additional 24 h. At harvest, DNA synthesis was measured as a function of [3H]thymidine incorporation; cell maturation was assessed by measuring alkaline phosphatase (ALPase) specific activity. Incorporation of [3H]uridine was used as a general indicator of RNA synthesis. Matrix protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and collagenase-nondigestible protein (NCP) as well as 35SO4 incorporation into proteoglycans. When RC cells were pretreated for 24 h with 24,25-(OH)2D3, they responded like RC cells that had received no pretreatment; further treatment of these cells with 1,25-(OH)2D3 had no effect on ALPase, proteoglycan, or NCP production, but CDP production was inhibited. However, when RC cells were pretreated for 36-120 h with 24,25-(OH)2D3, treatment with 1,25-(OH)2D3 caused a dose-dependent increase in ALPase, CDP, and proteoglycan synthesis, with no effect on NCP production. RC cells pretreated with 1,25-(OH)2D3 responded like RC cells that had not received any pretreatment. To determine whether these responses were specific to chondrocytes in the endochondral pathway, cells were isolated from the xiphoid process, a hyaline cartilage. In these cells, 1,25-(OH)2D3 inhibited ALPase, whereas 36 h of pretreatment with 24,25-(OH)2D3 caused these cells to lose their response to 1,25-(OH)2D3. 24,25-(OH)2D3 can directly regulate the differentiation and maturation of RC chondrocytes into GC chondrocytes, as evidenced by increased responsiveness to 1,25-(OH)2D3. 24,25-(OH)2D3 also promotes differentiation of cells derived from xiphoid cartilage, resulting in the loss of 1,25-(OH)2D3 responsiveness."

The 24,25-form tends to correlate with chondrogenesis whereas the 1,25 form tends to correlate with osteogenesis.  The 24,25-form downregulates it's own production in resting zone chondrocytes but upregulates the active form by growth chondrocytes.

Monocarboxylate transporter 10 functions as a thyroid hormone transporter in chondrocytes.

"untreated congenital hypothyroidism is marked by severe short stature. The monocarboxylate transporter 8 (MCT8) is a highly specific transporter for thyroid hormone. The hallmarks of Allan-Herndon-Dudley syndrome, caused by MCT8 mutations, are severe psychomotor retardation and elevated T(3) levels. However, growth is mostly normal. We therefore hypothesized that growth plate chondrocytes use transporters other than MCT8 for thyroid hormone uptake. Extensive analysis of thyroid hormone transporter mRNA expression in mouse chondrogenic ATDC5 cells revealed that monocarboxylate transporter 10 (Mct10) was most abundantly expressed among the transporters known to be highly specific for thyroid hormone, namely Mct8, Mct10, and organic anion transporter 1c1. Expression levels of Mct10 mRNA diminished with chondrocyte differentiation in these cells. Accordingly, Mct10 mRNA was expressed most abundantly in the growth plate resting zone chondrocytes in vivo. Small interfering RNA-mediated knockdown of Mct10 mRNA in ATDC5 cells decreased [(125)I]T(3) uptake up to 44% compared with negative control. Moreover, silencing Mct10 mRNA expression abolished the known effects of T(3), i.e. suppression of proliferation and enhancement of differentiation, in ATDC5 cells. Mct10 functions as a thyroid hormone transporter in chondrocytes and can explain at least in part why Allan-Herndon-Dudley syndrome patients do not exhibit significant growth impairment."

"TH inhibits proliferation and promotes differentiation of chondrocytes and is indispensable for normal growth"

"The SLC16A10 gene, which encodes MCT10, localizes to 6q21-q22 [and is associated with height growth]"

"in RZ chondrocytes, TH exerts its actions via TRα1."

"The thoracic VBGPs obtained from rats aged 1 day and 1, 4, 8, 16 and 28 weeks were identified using safranin O-fast green staining, and the height of the hypertrophic zone, proliferative zone, and resting zone were measured. The chondrocytes were isolated from these VBGPs with a modified trypsin-collagenase type II digestion method for primary culture in vitro. The expressions of proliferating cell nuclear antigen (PCNA) mRNA and protein was detected by real time-PCR and Western blotting, respectively.
The 1-day- and 1-week-old rats showed significantly greater hypertrophic zone and proliferative zone in the VBGPs than older rats; the proliferative zone was significantly greater in rats aged 4 weeks than in those aged 28 weeks. The resting zone was obviously greater in rats aged 1 day and 1 week than in older rats, and also greater in rats aged 4 weeks than in those aged 16 and 28 weeks. Obvious ossification in the resting zone occurred at 16 weeks, and most of the resting zone became ossified at 28 weeks. The expression of PCNA decreased at both the mRNA and protein levels as the rats grew.
The 3 zones of VBGPs are greater in rats aged 1 day and 1 week than in older ones. Ossification in the resting zone begins at 16 weeks, and till 28 weeks, most of the resting zone is ossified. The proliferation ability of VBGP chondrocytes decreases with the increase of age of the rats."

Study is in a foreign language unfortunately.

Distribution of type I and type II collagen gene expression during the development of human long bones.

"The temporal and spatial gene expression of collagen type I and type II during the development of the human long bones was studied by the technique of in situ hybridization covering the period from the cartilagenous bone anlage to the formation of a regular growth plate in the newborn. Analysis of the early stages around the seventh week of gestation revealed for type II collagen a strong hybridization signal limited to the chondrogenic tissue. The surrounding connective tissue and the perichondrium showed weak type I collagen expression, while the zones of desmal ossification like the clavicle gave a strong signal. Beginning with the eighth week of gestation, type I collagen mRNA was detectable in newly formed osteoblasts at the diaphysis and appeared along with the formation bone marrow, in the areas of enchondral ossification. Parallel to the development of the different zones of cartilage differentiation, a specific pattern of type II expression could be observed: type II was mainly found in the chondrocytes of the hypertrophic zone and to a lesser degree in the zone of proliferation, while the resting zone and the zone of provisional calcification showed little activity. This segregation of type II expression was most pronounced in the early stages of cartilage calcification and in the growth plate of the newborn."

"As prechondrogenic mesenchyme cells develop to chondrocytes, a dramatic increase in the cytoplasmatic volume, the rough endoplasmatic reticulum and the Golgi apparatus takes place. This is
paralleled by the switch from collagen type I, the predominant collagen of fibroblasts, to collagen type II, the major collagen found in cartilage"

"Limbs of human fetuses between the 7th and 15th menstrual weeks"  Mature chondrocytes never displayed Type I Collagen activity.  Type II collagen negative cells occur at the osteochondral junction.

"In embryonic limb development, FGF-4 stimulates Sonic hedgehog (Shh) expression in a positive feedback loop that coordinates proximal-distal and anterior-posterior patterning of the cartilaginous anlagen"

According to the paper 20-50% of cells in the bone marrow have the ability to differentiate into chondrocytes.

"progenitor cells with chondrogenic capacity have been isolated from the superficial zone of articular cartilage"

"chondroprogenitors have been identified in arthritic cartilage after their migration from the bone marrow through breaks in the tidemark and into the diseased cartilage"<-meaning chondroprogenitors exist in the bone marrow.

Some great diagrams in this paper.

"When the cells aggregate, MCs[mesenchymal cells] begin to produce collagen I, fibronectin, and proteoglycans. The result of the strong interactions that cells establish with their environment is the formation of a dense mass of MCs that immediately begins to differentiate into chondroblasts. Condensed MCs start expressing mainly the transcription factor Sox9 that controls downstream genes involved in chondrogenesis, promoting these progenitor cells to secrete cartilage-specific ECM molecules"

"MMP1 and MMP2 have the capacity to degrade cartilage matrix, and they are characterized as the MMPs that are involved in earlier chondrogenesis. Specifically, blockage of MMP2 function supports precartilage condensation and chondrogenesis, and MMP1 knockout mice show decreased chondrocyte proliferation in the proliferative zone of the growth plates of long bones." MMP2 is increased in LSJL so perhaps we should find a way to decrease it's expression.

"overexpression of human Sox9 in murine ESCs (mESCs) leads to upregulated expression of the cartilage markers collagen IIA, aggrecan, and pax1 even in undifferentiated ESCs"

"fibroblasts can undergo spontaneous chondrogenesis in simple three-dimensional culture conditions"


  1. the growth plate sits above the emphasis so should we be clamping higher up or as low as we can.

  2. Could you please answer my question ^^