Monday, March 5, 2012

Gene expression of normal growth plate

Microarray analysis of perichondral and reserve growth plate zones identifies differential gene expressions and signal pathways.

"Normal Sprague-Dawley rat growth plate chondrocytes from the perichondral zone (PC)[the layer of fibrous connective tissue that envelops cartilage] and reserve zone (RZ) were isolated by laser microdissection and then subjected to microarray analysis. In order to most comprehensively capture the unique features of the two zones, we analyzed both the most highly expressed genes and those that were most significantly different from the proliferative zone (PZ) as a single comparator. Confirmation of the differential expression of selected genes was done by quantitative real-time RT-PCR. A total of 8 transcripts showing high expression unique to the PC included translationally-controlled tumor protein (Tpt1), connective tissue growth factor (Ctgf), mortality factor 4 (Morf4l1), growth arrest specific 6 (Gas6), type V procollagen (Col5a2){upregulated by LSJL}, frizzled-related protein (Frzb), GDP-dissociation inhibitor 2 (Gdi2) and Jun D proto-oncogene (Jund). In contrast, 8 transcripts showing unique high expression in the RZ included hyaluronan and proteoglycan link protein 1 (Hapln1){upregulated by LSJL}, hemoglobin beta-2 subunit, type I procollagen (Col1a2), retinoblastoma binding protein 4 (LOC685491), Sparc-related modular calcium binding 2 (Smoc2) {Upregulated by LSJL}, and calpastatin (Cast). Other genes were highly expressed in cells from both PC and RZ zones, including collagen 2a1, collagen 9a3 {both upregulated by LSJL}, ND3, catenin (cadherin associated protein) beta 1, Eef1a1{Eef1a1 was downregulated by LSJL}, HGMB1, Rpl35a, Mtap7, Rcn1, Thbs1, Rsp6, Cpe, Cpt1a, Sparc, plexin B2 (Plxnb2), and gGja1. Functional classification of the most highly expressed transcripts were analyzed, and the pathway analysis indicated that ossification, bone remodeling, and cartilage development were uniquely enriched in the PC whereas both the PC and RZ showed pathway enrichment for skeletal development, extracellular matrix structural constituent, proteinaceous extracellular matrix, collagen, extracellular matrix, and extracellular matrix part pathways."

"RNAs highly expressed within both the PC and RZ were 21 genes, including RGD 1308977 (similar to RIKEN cDNA), RGD 1306734 (similar to hypothetical protein FLJ32743), and LOC 685491 (similar to retinoblastoma binding protein 4), Mgp (matrix Gla protein)."

"Col9a3 may contribute to the three-dimensional integrated structure of type II collagen molecules"

"Matrix GLA protein (Mgp) is a mineral binding extracellular matrix protein synthesized by growth plate cartilage chondrocytes"

"Reticulocalbin (Rcn1), which is distributed predominantly in endocrine and exocrine organs, is one member of the Ca2+-binding proteins in the secretory pathway "

"Ctgf deficiency leads to skeletal dysmorphisms as a result of impaired chondrocyte proliferation and extracellular matrix composition within the hypertrophic zone"

"Frzb-1 [is] strong in prehypertrophic chondrocytes."

Spatial and temporal regulation of gene expression in the mammalian growth plate.

"we used microdissection to collect individual growth plate zones from proximal tibiae of 1-week rats and the PZ and early hypertrophic zones of growth plates from 3-, 6-, 9-, and 12-week rats"

"candidate markers for the resting zone [include Sfrp5], bone morphogenetic protein 3 (Bmp3) and gremlin 1 (Grem1) two genes involved in BMP signaling, epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (Efemp1), and collagen type 14 alpha 1 (Col14a){up in LSJL}."

Other Resting Zone Markers:

Percentage of total upregulated in LSJL: 17.5%
Downregulated: 5%

Proliferating Zone Markers:

Hypertrophic Zone:
Cmkor1{up as Cxcr7}
Col6A2{up and down, counted twice in percentages}
Col10A1(up in LSJL)
IBSP(up in LSJL as BSP)
Igsf4a{down as Cadm1}

Percentage of total upregulated by LSJL: 11.6%
Downregulated: 5.4%

Genes upregulated with youth:

Percentage upregulated of total: 4.76%
Downregulated: 2.38%

Genes upregulated with age:
Col6a2{up and down}

Upregulated: 18%
Downregulated: 4.16%

"Some important transcription factors were being significantly regulated between the resting and proliferative zones (Dlx5, c-Fos{up in LSJL}, c-Maf, Nrf2, Runx2, Runx3, Sox6, upregulation; Hif1α, Shox2, downregulation), or between the proliferative and hypertrophic zones (Atf3{up}, c-Fos{up}, c-Maf, MafB{down}, Mef2c, Mef2d, Runx2, Sox6, upregulation; Hif1α, Runx3, Shox2, downregulation)"

"several transcription factors showed significant upregulation (Hif1α, Jun{up in LSJL}) or downregulation (Mef2c, Runx2, Runx3, Sox9{up in LSJL}) with age."

"Gli3{up in LSJL} represses the transition from round into columnar chondrocytes"

Temporal and spatial expression of a growth-regulated network of imprinted genes in growth plate.

"We characterized expression of the network during postnatal growth in microdissected metaphyseal bone and growth plate zones of 1-, 3-, and 9-week-old rats using real-time PCR. The expression pattern of the network is modified in growth plate. Similar to the coordinated decline previously observed in kidney, lung, liver, and heart, expression of all genes, except Gtl2, decreased with age in metaphyseal bone. On the contrary, Mest, Dlk1, H19{up}, and Gtl2 decreased, and Cdkn1c, Grb10, and Slc38a4{up} increased with age in growth plate. During differentiation from resting to hypertrophic zone, Mest, Dlk1, Grb10, and Gtl2 expression decreased, whereas Slc38a4 expression increased. In particular, developmental changes in the expression of growth-promoting genes, Mest, Dlk1, Gtl2, and growth-inhibitory genes, Cdkn1c, [Ndn], and Grb10, may contribute to the decline in longitudinal bone growth that occurs with age."

"Targeted ablation of either Dlk1, Mest, Gtl2, Igf2, Plagl1, or Peg3 results in reduced body size at birth or earlier"

"Dlk1, Mest, Gtl2, and Igf2 ablation also negatively affect postnatal growth"

"Disruption of Grb10 in mice results in overgrowth of mutant embryos"

Expression profiling of human fetal growth plate cartilage by EST sequencing.

"Here, we describe the sequences of 4,748 clones from a human growth plate cartilage cDNA library generated from 20 weeks prenatal-2 years postnatal specimens. In silico analysis of these sequences revealed 1,688 individual transcription units, corresponding to known (1,274) and to novel, yet uncharacterised potential genes (414). The tissue specificity of the library was reflected by its corresponding EST profile representing a total of approximately 10% proteins already shown to be involved in cartilage/bone development or homeostasis. The EST profile also reflects the developmental stage of the tissue with significant differences in the expression of matrix proteins compared to corresponding EST profiles from 8-12 and 12-20 week human fetal cartilage."

"As expected, the most prevalent transcript encodes the type II procollagen (COL2A1) with 103 ESTs (56,6% of collagen clones). The next most highly expressed transcripts corresponded to COL9A1 with 15 ESTs (8.24%), COL9A3 with 12 ESTs (6.59%), COL11A2 with 9 ESTs (4.95%), and COL9A2 with 4.47% of the collagen, while COL6A2, COL16A1 and COL3A1 represented 2.75%, 2.2% and 1.65%, respectively. The remaining identified collagen transcripts are represented by less than 3 ESTs (< 1.5% of the total collagen clones). It has to be emphasized that the number of ESTs for the different chains of collagen I and IX does not reflect the expected molar ratios of COL1A1 / COL1A2 (2 : 1) and COL9A1 / COL9A2 / COL9A3 (1 : 1 : 1), respectively"

"collagen XVI is strongly expressed in differentiating chondrocytes, where it is a component of small heterotypic D-banded fibrils, mainly occurring in the territorial matrix of chondrocytes. Based on in vitro studies, COL16A1 has been suggested to play a role in later chondrogenesis, which would match with the present data."<-Col16a1 is upregulated in LSJL.

"transcripts encoding 11 different individual proteoglycans were identified (38 ESTs), some of them with high expression levels, like biglycan (BGN) with 9 ESTs, fibromodulin (FMOD), chondroitin sulfate proteoglycan 4 (CSPG4) and aggrecan 1 (AGC1) with 6 ESTs."<-All but FMOD were upregulated in LSJL.

Syndecan 4{up} and 2{down} also had significant EST transcripts.  HAPLN1 another EST significantly observed was upregulated in LSJL.

Non-Collagen ESTs observed altered by LSJL:

" transcripts of seven different matrix-metabolising genes were identified (64 ESTs). The most prevalent transcript in this group was matrix metalloproteinase-3 (MMP3) with 34 ESTs, which encodes a proteoglycanase, followed by MMP1 with 22 ESTs and MMP13 with 4 ESTs, both representing collagenases. MMP3 and MMP13 were also reported to be the most strongly expressed MMPs in 12–20 week fetal cartilage."<-MMP3 was upregulated by 3.6 fold.

Profiling genes expressed in human fetal cartilage using 13,155 expressed sequence tags.

8-12 week old fetal cartilage.

Genes expressed whose expression was altered in LSJL:
HTRA1(as PRSS11){up}
TIMP1{up very highly}

"The fact that IGF-II was the most abundant growth factor identified in fetal cartilage suggests that both glypican 3 and IGF-II are important in fetal cartilage development."

Gene expression during endochondral bone development: Evidence for coordinate expression of transforming growth factor β1 and collagen type I

"Subcutaneous implatation of demineralized bone particles (DBP) into rats induces the formation of a bone ossicle by a tightly controlled sequence of chondro- and osteo-inductive events which are directly comparable to those which occur in normal endochondral bone development.  To examine the expression of genes in this system, RNA was isolated from implants every 2 days over a time course spanning 3 to 19 days after implantation of DBP into rats. Cellular levels of mRNA transcripts of cell-growth-regulated and tissue-specific genes were examined by slot blot analysis and compared to the morphological changes occuring during formation of the ossicle. Analysis of the mRNA levels of histone H4 and c-myc, markers of proliferative activity, revealed several periods of actively proliferating cells, corresponding to (1) production of fibroprogenitor cells (day 3), (2) onset of bone formation (day 9), and (3) formation of bone marrow (day 19). The mRNA levels of collagen type II, a phenotypic marker of cartilage, peaked between days 7 and 9 post-implantation, corresponding to the appearance of chondrocytes in the implant, and rapidly declined on day 11 (to 5% of maximum value) when bone formation was observed. The peak mRNA levels of collagen type I, found in fibroblasts and osteoblasts, occurred first with the onset of bone formation (days 7–10) and again during formation of bone marrow (day 19). This study has demonstrated that the temporal patterns of mRNA expression of cartilage type II and bone type I collagens coincide with the morphological sequence in this model of endochondral bone formation. Further, the mRNA levels of transforming growth factor β1 (TGFβ) were compared to those of collagen types I and II; a direct temporal correlation of TGFβ mRNA levels with that of collagen type I was found throughout the developmental time course. This observation of a tightly coupled relationship between TGFβ and type I collagen mRNA levels is consistent with a functional role for TGFβ in extracellular matrix production during in vivo bone formation."

"The time points in the present study were selected to illustrate the progressive stages of endochondral bone formation in the DBP implants. By day 3 the early stage of recruitment of fibroprogenitor cells around the particles of the implant was apparent. By day 7 chondroblasts were observed in the areas adjacent to the bone particles. On day 9, more of the implant had undergone chondrogenesis and the cartilage matrix had mineralized.  Bone formation, noted by the presence of osteoblasts and newly formed osteoid, was observed on day 11. Completion of bone formation and the onset of the bone remodeling stage were observed by day 15, and marrow formation and hematopoeisis were evident at day 18. Thus, the DBP implants exhibited the chondro- and osteogenic events which normally occur during endochondral bone formation"

Histone H4, c-Myc, Col1a1, TGFB, and Col2a1 increased in expression.

"Bone powder was prepared from long bones of adult rats (8-10 weeks). The diaphyses of the long bones were cleaned, pulverized in liquid nitrogen, sieved to particle size of 75-250 pm, and then acid demineralized. Fifty milligrams of DBP was implanted in bilateral subcutaneous pockets on the thoracic region in 28-day-old male Sprague Dawley CD strain rats"

No comments:

Post a Comment