Chondrocyte-specific transcription factors

This post will be confusing to most and normally I would bury it in the blog for reference purposes but I'm hoping that a transcription factor expert could look these over and find any evidence that LSJL induces ectopic chondroinduction.

Chondrogenic transcription factors up in LSJL:

Sox9

GATA4

TRPS1{downstream of GDF5}

Barx2{downstream of GDF5}
Gli3
Prrx1
Prrx2
Pax1
Lhx8

It's predicted that GDF5 is involved in LSJL.

Other transcription factors involved in LSJL.

Up:
Fos
ATF3
Meox2
Hey2
Hoxd10
WWTR1
Dbx2
Hoxb1
Jun
Phox2b

Down(bold are related to chondrogenesis):
E2F6
Smad1
Gtf2i
Npas4
Dbx1
Cdx1
Foxb1
HNF4A
Hoxb13
Tcf7{According to Regulation of Tcf7 by Runx2 in chondrocyte maturation and proliferation. knockout of Tcf7 causes dwarfism}
Tcf12
Mxd4{According to Novel late response genes of PTHrP in chondrocytes., Mxd4 is downregulated by PTHrP}
Btf3
Mafb
Nrf1{Mentioned at a growth plate meeting: Meeting Report:The 7th ESPE Growth Plate Working Group Symposium - EUROGROP June 27th 2007, Helsinki, Finland. but I cannot get the details}
Tcfec
Mxd3
Tfdp2
E2F1

Placenta to cartilage: direct conversion of human placenta to chondrocytes with transformation by defined factors.

"mouse and human fibroblasts can be reprogrammed to a pluripotent[iPSCs] state with a combination of four transcription factors. We hypothesized that combinatorial expression of chondrocyte-specific transcription factors could directly convert human placental cells into chondrocytes. Starting from a pool of candidate genes, we identified a combination of only five genes (5F pool)-BCL6, T (also called BRACHYURY), c-MYC, MITF, and BAF60C (also called SMARCD3)-that rapidly and efficiently convert postnatal human chorion and decidual cells into chondrocytes. The cells generated expressed multiple cartilage-specific genes, such as Collagen type II α1, LINK PROTEIN-1, and AGGRECAN, and exhibited characteristics of cartilage both in vivo and in vitro. Expression of the endogenous genes for T and MITF was initiated, implying that the cell conversion is due to not only the forced expression of the transgenes, but also to cellular reprogramming by the transgenes."

MyoD is the gene responsible for transdifferentiation into different cell types.

"Murine chondrocytes can be converted from fetal fibroblasts by the direct reprogramming method using the cartilage-specific transcription factors Sox9, c-Myc, and Klf4"

"expression of placenta-associated genes such as GATA3, CD200{CD200r3 is downregulated by LSJL}, PDCD1LG2, OLR1, TEK, HSD17B2, and FOXF1 was lost in the infected cells{chondrogenic differentiation induced cells}."

"T{BRACHYURY} is necessary for chondrogenic conversion"

"c-Myc is a cell cycle driver, and Klf4 is involved in the down-regulation of p53"

"T and MITF may act as inducers of chondrogenic fate determinant, and BAF60C may act as epigenetic modifier"

BAF60C may permit binding of GATA4{up in LSJL} to chondrocytes.

Identification of a 3Kbp Mechanoresponsive Promoter Region in the Human Cartilage Oligomeric Matrix Protein Gene.

"the proximal 3 Kb of the human cartilage oligomeric matrix protein promoter is sufficient to mediate a mechanoresponse in human articular chondrocytes and stem cells, and that the magnitude of mechanoresponse correlates to the regulation of the endogenous gene at the RNA and protein level."

"the growth plate aligns itself with the axis of compressive mechanical force."

"there was no difference in the viability of chondrocytes or BMSC subject to 5 days of dynamic compression and their respective uncompressed controls "

FoxA family members are crucial regulators of the hypertrophic chondrocyte differentiation program.

"FoxA factors are induced during chondrogenesis, bind to conserved binding sites in the collagen X enhancer, and can promote the expression of a collagen X-luciferase reporter in both chondrocytes and fibroblasts. In addition, we demonstrate by both gain- and loss-of-function analyses that FoxA factors play a crucial role in driving the expression of both endogenous collagen X and other hypertrophic chondrocyte-specific genes. Mice engineered to lack expression of both FoxA2 and FoxA3 in their chondrocytes display defects in chondrocyte hypertrophy, alkaline phosphatase expression, and mineralization in their sternebrae and, in addition, exhibit postnatal dwarfism that is coupled to significantly decreased expression of both collagen X and MMP13 in their growth plates."

"Runx2 and Runx3 are expressed in chondrocytes as they initiate differentiation, and loss of these factors (in genetically engineered mice) severely delays or blocks chondrocyte hypertrophy in a number of developing bones"

"ectopic expression of Runx2 in immature chondrocytes drives premature cellular maturation and induces expression of collagen X and other hypertrophic markers"

MEF2C and MEF2D{down in LSJL} promote Runx2 expression.

"FoxA2{activates Col10 which was up in LSJL} and FoxA3 are expressed in developing cartilages"

" FoxA factors, which are present in chondrocytes yet absent from fibroblasts, may confer competence for Runx2, Smad1{down}, and MEF2C to activate expression of the collagen X luciferase reporter in the former cell type."

"FoxA3−/− mice were smaller, with a specific deficiency of cells in the hypertrophic zone but not in the proliferating zone "

"both FoxA2 and FoxA3 are necessary to promote high-level expression of several hypertrophic chondrocyte markers in the growth plate, including collagen X, alkaline phosphatase, and MMP13."

"Sox transcription factors, which are expressed in immature chondrocytes and downregulated in hypertrophic chondrocytes, may act to compete for FoxA factor binding to the collagen X enhancer and thereby inhibit precocious expression of collagen X and/or other hypertrophic markers in immature chondrocytes. Indeed, others have noted that mutation of a Sox9 binding site within the murine Col10a1 enhancer is required to repress expression of a transgene driven by this enhancer in nonhypertrophic chondrocytes "

Control of mesenchymal lineage progression by microRNAs targeting skeletal gene regulators Trps1 and Runx2.

"seven RUNX2-targeting miRNAs (miR-23a, miR-30c, miR-34c, miR-133a, miR-135a, miR-205, and miR-217) also regulate the chondrogenic GATA transcription factor tricho-rhino-phalangeal syndrome I (TRPS1){up}. Although the efficacy of each miRNA to target RUNX2 or TRPS1 differs in osteoblasts and chondrocytes, each effectively blocks maturation of precommitted osteoblasts and chondrocytes. Furthermore, these miRNAs can redirect mesenchymal stem cells into adipogenic cell fate with concomitant up-regulation of key lineage-specific transcription factors."

TRPS1 is a Zn finger transcription factor that promotes chondrogenesis.

"five RUNX2-targeting miRNAs potentially target the Kruppel-like transcription factor KLF12, a principal transcriptional repressor of the activator protein-2 α (AP-2 α) gene, which controls vertebrate development"

"miR-30c dramatically up-regulates expression of early-stage ECM genes when administered prior to differentiation of MC3T3 osteoblasts, including three collagen genes (Col1A1, Col1A2, and Col3A1) and matrix metalloproteinases 2 and 9 (Mmp2 and Mmp9). In addition, late-stage ECM genes, including OP, osteocalcin (OC-BGLAP), and Dmp1 expression, are suppressed by miR-30c"

"miR-30c modulates expression of genes encoding ligands and receptors of TGFβ, FGF, and IGF signaling and that this modulation is strikingly different during differentiation in ATDC5 chondrocytes and MC3T3 osteoblasts"

"TGFβs, TGFβRs, and SMADs are clearly down-regulated by miR-30c in chondrocytes but up-regulated in osteoblasts. Also, expression of genes for Fgf2, Fgfr1, Fgfr2, and Igf1r is clearly stimulated in MC3T3 cells, whereas expression of Fgf1, Fgf3, Fgfr1, and Igf1 genes is suppressed in ATDC5 cells."<-Despite the presence of TRPS1, FGF2 and FGFR1 were upregulated.

Trps1 plays a pivotal role downstream of Gdf5 signaling in promoting chondrogenesis and apoptosis of ATDC5 cells.

"Tricho-rhino-phalangeal syndrome (TRPS) is an autosomal dominant skeletal disorder caused by mutations of TRPS1. Based on the similar expression patterns of Trps1 and Gdf5, we hypothesized a possible functional interaction between these two molecules. Using a chondrogenic cell line (ATDC5), we investigated the association of Gdf5-mediated signaling pathways with Trps1 and the phenotypic changes of ATDC5 cells due to over-expression or suppression of Trps1. Treatment of cells with Gdf5 enhanced Trps1 protein levels and phosphorylation of p38 mitogen-activated protein kinase (MAPK) in a dose-dependent manner. Nuclear translocation of Trps1 was also induced by Gdf5. These effects were blocked by a dominant negative form of activin-linked kinase 6 (dn-Alk6) and by SB203580, an inhibitor of the p38 MAPK pathway. Conversely, Gdf5 expression was suppressed by the over-expression of Trps1. Trps1-overexpressing ATDC5 (O/E) cells differentiated into chondrocytes more quickly than mock-infected control cells, whereas cells transfected with dn-Alk6 showed slower differentiation. On the other hand, O/E cells showed an increase of apoptosis along with the up-regulation of cleaved caspase 3 and down-regulation of Bcl-2, whereas dn-Alk6 cells showed suppression of apoptosis. In conclusion, Trps1 acts downstream of the Gdf5 signaling pathway and promotes the differentiation and apoptosis of ATDC5 cells."

"Trps1 is a transcriptional repressor, which interacts with the dynein light chain and RING finger protein 4 (Rnf4) in the nucleus"

"Trps1 is mainly expressed in the joints and in the limb growth plate cartilages during late embryogenesis. Mice with Trps1 deficiency die of respiratory failure soon after birth and show changes such as skeletal deformities that resemble those seen in human TRPS patients"

"Expression of Trps1 by ATDC5 cells was not observed in the absence of insulin, while it gradually increased in the presence of insulin "

"Trps1 mRNA levels were not significantly enhanced by Gdf5 compared with Trps1 protein levels"

"expression of Bcl-2 mRNA was significantly decreased by the over-expression of Trps1, whereas transfection with dn-Alk6 resulted in elevated Bcl-2 expression"

"enhancement of apoptosis in Trps1-overexpressing cells"

"Trps1 might negatively regulate a transcriptional repressor of the type II and type X collagen genes. It has been reported that the other transcription factor downstream of Gdf5, Barx2{up in LSJL}, binds to the intronic regulatory region of the Col2a1 gene during chondrogenesis. We found that Trps1 binds to two GATA binding sites in the promoter of the Stat3 gene and represses the expression of Stat3"

Trps1, a regulator of chondrocyte proliferation and differentiation, interacts with the activator form of Gli3.

"Trps1 interacts with Indian hedgehog (Ihh)/Gli3 signaling and regulates chondrocyte differentiation and proliferation. We demonstrate that Trps1 specifically binds to the transactivation domain of Gli3{up in LSJL} in vitro and in vivo, whereas the repressor form of Gli3 does not interact with Trps1. A domain of 185aa within Trps1, containing three predicted zinc fingers, is sufficient for interaction with Gli3. Using different mouse models we find that in distal chondrocytes Trps1 and the repressor activity of Gli3 are required to expand distal cells and locate the expression domain of Parathyroid hormone related peptide. In columnar proliferating chondrocytes Trps1 and Ihh/Gli3 have an activating function. The differentiation of columnar and hypertrophic chondrocytes is supported by Trps1 independent of Gli3. Trps1 seems thus to organize chondrocyte differentiation interacting with different subsets of co-factors in distinct cell types."

"In the presence of Hh signals, Gli2 and Gli3 are thought to enter the nucleus to activate target genes, whereas in absence of Hh signals, Gli3 and, to a lesser extend, Gli2 are proteolytically processed into a short, N-terminal repressor form that inhibits the expression of Hh target genes"<-LSJL increases Dhh signaling.

"Measurements of Trps1−/− ulnae revealed a reduction to 80% in length and 82% in diameter"  Gli3 knockout decreases height as well.

"The zone of proliferating chondrocytes can further be subdivided into distal chondrocytes, which express Fibroblast growth factor receptor 1 (Fgfr1){up} and Unique cartilage matrix-associated protein (Ucma), and columnar chondrocytes, which are characterized by the expression of Bone Morphogenetic Protein 7 (Bmp7), Fgfr3 and the lack of Ihh expression"

"in absence of Ihh signals Gli3 acts as a strong repressor of proliferation"

Trps1 regulates proliferation and apoptosis of chondrocytes through Stat3 signaling.

"disrupted Trps1 gene develop a chondrodysplasia characterized by diminished chondrocyte proliferation and decreased apoptosis in growth plates. Our analyses revealed that Trps1 is a repressor of Stat3 expression, which in turn controls chondrocyte proliferation and survival by regulating the expression of cyclin D1 and Bcl2. Our conclusion is supported (i) by siRNA-mediated depletion of Stat3 in Trps1-deficient chondrocytes, which normalized the expression of cyclin D1 and Bcl2, (ii) by overexpression of Trps1 in ATDC5 chondrocytes, which diminished Stat3 levels and increased proliferation and apoptosis, and (iii) by mutational analysis of the GATA-binding sites in the Stat3 gene, which revealed that their integrity is critical for the direct association with Trps1 and for Trps1-mediated repression of Stat3."

"a strong reduction in cyclin D1 expression in freshly isolated Trps1−/− chondrocytes"

The homeobox transcription factor Barx2 regulates chondrogenesis during limb development.

"Barx2 is necessary for mesenchymal aggregation and chondrogenic differentiation. In accordance with these findings, Barx2 regulates the expression of several genes encoding cell-adhesion molecules and extracellular matrix proteins, including NCAM and collagen II (Col2a1) in the limb bud. Barx2 bound to elements within the cartilage-specific Col2a1 enhancer, and this binding was reduced by addition of Barx2 or Sox9 antibodies, or by mutation of a HMG box adjacent to the Barx2-binding element, suggesting cooperation between Barx2 and Sox proteins. Moreover, both Barx2 and Sox9 occupy Col2a1 enhancer during chondrogenesis in vivo. We also found that two members of the BMP family that are crucial for chondrogenesis, GDF5 and BMP4, regulate the pattern of Barx2 expression in developing limbs."

"Dlx2 acts downstream of BMP signal"

"Barx2 is involved in regulation of Ca+2-independent and Ca+2-dependant adhesion."

"Barx2 constructs induced chondrogenesis in micromass cultures prepared from the proximal region of the limb bud, whereas micromass cultures made from the distal mesenchyme of the same limb bud showed a fivefold increase in chondrogenesis after overexpression of Barx2. These results suggest that Barx2 can promote chondrogenesis only in the distal mesenchyme of the limb bud, which contains mostly uncommitted cells "

"Barx2 and GDF5 are co-expressed during limb development"

"application of GDF5 or BMP4-soaked beads induced ectopic expression of Barx2 all around the bead"

"Barx2 is also necessary for chondrogenesis and interacts functionally with both BMPs and Sox9."

" GDF5 can induce chondrogenesis in mesenchymal cells that have not yet condensed, while BMPs induce chondrogenic differentiation only after condensation"

Hdac-mediated control of endochondral and intramembranous ossification.

"Several Hdacs contribute to the molecular pathways and chromatin changes that regulate tissue-specific gene expression during chondrocyte and osteoblast specification, maturation, and terminal differentiation. In this review, we summarize the roles of class I and class II Hdacs in chondrocytes and osteoblasts. The effects of small molecule Hdac inhibitors on the skeleton are also discussed."

"Hdac1 associates with Nkx3.2, an essential transcriptional repressor governing the formation of the cartilage anlagen during endochondral ossification. Furthermore, Hdac1 represses Smad-dependent signaling to control responses to Bmp2, a critical chondrogenic factor.29 Hdac4 and Hdac5 also regulate Smad signaling downstream of Tgf-β and thus may participate in controlling the process of chondrogenesis."

"Hdac4 binds to Runx2 and represses Runx2-induced hypertrophy"

Directed differentiation of human embryonic stem cells toward chondrocytes.

GATA4, Sox9, and KDR are up in LSJL.

The role of NELL-1, a growth factor associated with craniosynostosis, in promoting bone regeneration.

"NELL-1, which is a potent growth factor that is highly specific to the osteochondral lineage, and has demonstrated robust induction of bone in multiple in vivo models from rodents to pre-clinical large animals. NELL-1 is preferentially expressed in osteoblasts under direct transcriptional control of Runx2, and is well-regulated during skeletal development. NELL-1/Nell-1 can promote orthotopic bone regeneration via either intramembranous or endochondral ossification, both within and outside of the craniofacial complex. Unlike BMP-2, Nell-1 cannot initiate ectopic bone formation in muscle, but can induce bone marrow stromal cells (BMSCs) to form bone in a mouse muscle pouch model, exhibiting specificity that BMPs lack. In addition, synergistic osteogenic effects of Nell-1 and BMP combotherapy have been observed, and are likely due to distinct differences in their signaling pathways. NELL-1's unique role as a novel osteoinductive growth factor makes it an attractive alternative with promise for future clinical applications. [Note: NELL-1 and NELL-1 indicate the human gene and protein, respectively; Nell-1 and Nell-1 indicate the mouse gene and protein, respectively.]"

" A 100 ng/mL quantity of NELL-1 protein can significantly activate the ERK1/2 and JNK1/2/3 kinases in primary rat fetal calvarial (RFC) cells after only 10 min of stimulation"

TRPS1

TRPS1 is upregulated by LSJL.

ROLE OF TRPS1 IN ENDOCHONDRAL BONE FORMATION

"Mutations of the human TRPS1 gene cause a dominantly inherited skeletal dysplasia tricho-rhino-phalangeal syndrome (TRPS). Although the gene was identified several years ago, the molecular and cellular mechanisms underlying TRPS are largely unknown. Trps1 is a GATA-type transcription factor that acts as a transcriptional repressor. Recently, we have demonstrated that disruption of the Trps1 gene in mice results in dramatic elongation of growth plates and delayed replacement of cartilage by bone. These abnormalities are accompanied by increased Indian hedgehog (Ihh) expression and elevated Ihh and BMP signaling. Although BMP and Ihh pathways act in parallel to regulate various aspects of endochondral bone formation, it is unclear how they are integrated and controlled at the transcriptional level. The hypothesis of this proposal is that Trps1 constrains the Ihh-BMP positive feedback loop to assure timely progression of chondrocyte maturation and synchronization of chondrocyte development with perichondrial mineralization. We propose studies aimed at elucidating the Trps1 molecular network and its role in regulation of the cross-talk between differentiating chondrocytes and perichondrium. Specifically, we will focus on understanding the mechanisms of delayed cartilage removal and endochondral ossification in Trps1 mutant mice. To address these questions we propose the following specific aims: 1. To understand the molecular and cellular mechanisms of the growth plate elongation caused by disruption of the Trps1 gene. 2. To determine how Trps1 regulates BMP and hedgehog signaling. The first aim will be accomplished by histological and molecular analyses of abnormalities in the growth plate of the mouse model of TRPS. To achieve the second aim we will employ a combination of electrophoretic mobility shift assay (EMSA), reporter expression assays and analyses of the effect of the Trps1 deficiency and over-expression on BMP and hedgehog signaling in a cellular model of chondrogenesis. Additionally, we will test the Trps1 and Ihh genetic antagonism using Trps1;Ihh double mutant mice. Results of the proposed studies will define molecular mechanisms underlying skeletal dysplasia in tricho-rhino-phalangeal syndrome. Importantly, the results of these studies will directly impact our understanding of the transcriptional control of BMP and hedgehog signaling, that are widely involved in the development of multiple organ systems."

Here's the study noted:

Uncoupling of chondrocyte differentiation and perichondrial mineralization underlies the skeletal dysplasia in tricho-rhino-phalangeal syndrome.

"Patients with TRPS have short stature, hip abnormalities, cone-shaped epiphyses and premature closure of growth plates reflecting defects in endochondral ossification. The TRPS1 gene encodes for the transcription factor TRPS1 that has been demonstrated to repress transcription in vitro. To elucidate the molecular mechanisms underlying skeletal abnormalities in TRPS, we analyzed Trps1 mutant mice (Trps1DeltaGT mice[involves a frame deletion of the binding element of Trps1]). Analyses of growth plates demonstrated delayed chondrocyte differentiation and accelerated mineralization of perichondrium in Trps1 mutant mice. These abnormalities were accompanied by increased Runx2 and Ihh expression and increased Indian hedgehog signaling. We demonstrated that Trps1 physically interacts with Runx2 and represses Runx2-mediated trans-activation. Importantly, generation of Trps1(DeltaGT/+);Runx2(+/-) double heterozygous mice rescued the opposite growth plate phenotypes of single mutants, demonstrating the genetic interaction between Trps1 and Runx2 transcription factors. Collectively, these data suggest that skeletal dysplasia in TRPS is caused by dysregulation of chondrocyte and perichondrium development partially due to loss of Trps1 repression of Runx2."

"Trps1 is highly expressed in regions where Runx2 is downregulated"

"in Trps1ΔGT/ΔGT mice, there is a larger zone of cells co-expressing both Ihh and Col10a1[in addition to expanded growth plates], which indicates an increased number of cells in the transition from prehypertrophic to mature hypertrophic stage."  However height remained largely the same.

"Runx2 expression in perichondrium inhibits chondrocytes proliferation through Fgf18"

The multi zinc-finger protein Trps1 acts as a regulator of histone deacetylation during mitosis.

"TRPS1, the gene mutated in human "Tricho-Rhino-Phalangeal syndrome," encodes a multi zinc-finger nuclear regulator of chondrocyte proliferation and differentiation. Trps1 [controls] mitotic progression in chondrocytes. Loss of Trps1 in mice leads to an increased proportion of cells arrested in mitosis and, subsequently, to chromosome segregation defects. Trps1 acts as regulator of histone deacetylation. Trps1 interacts with two histone deacetylases, Hdac1 and Hdac4, thereby increasing their activity. Loss of Trps1 results in histone H3 hyperacetylation, which is maintained during mitosis. Consequently, chromatin condensation and binding of HP1 is impaired, and Trps1-deficient chondrocytes accumulate in prometaphase. Overexpression of Hdac4 rescues the mitotic defect of Trps1-deficient chondrocytes, identifying Trps1 as an important regulator of chromatin deacetylation during mitosis in chondrocytes. The control of mitosis can be linked to the regulation of chondrocyte differentiation by epigenetic consequences of altered Hdac activity. "

DNA condensation requires histone deacetylases.  Cells with impaired Hdac function are defective.  TRPS1 knockout decreases cellular proliferation.

Addition of Hdac4 didn't affect the proportion of cells in the G2/M phase in the TRPS1+/+ group in contrast to the knockout group where addition of HDAC4 decreased portion of cells in G2/M phase.  Thus meaning that overexpression of TRPS1 may not cause skeletal overgrowth.  Addition of Hdac4 did alter some H3K9 acetylation parameters however.