Andreia Ionescu

PTHrP Modulates Chondrocyte Differentiation through AP-1 and CREB Signaling

During the process of differentiation, chondrocytes integrate a complex array of signals from local or systemic factors like parathyroid hormone-related peptide (PTHrP), Indian hedgehog, bone morphogenetic proteins and transforming growth factor β. While PTHrP is known to be a critical regulator of chondrocyte proliferation and differentiation, the signaling pathways through which this factor acts remain to be elucidated. Here we show that both cAMP response element-binding protein (CREB) and AP-1 activation are critical to PTHrP signaling in chondrocytes. PTHrP treatment leads to rapid CREB phosphorylation and activation, while CREB DNA binding activity is constitutive. In contrast, PTHrP induces AP-1 DNA binding activity through induction of c-Fos protein expression. PTHrP activates CRE and TRE reporter constructs primarily through PKA-mediated signaling events. Both signaling pathways were found to be important mediators of PTHrP effects on chondrocyte phenotype. Alone, PTHrP suppresses maturation and stimulates proliferation of the chondrocyte cultures. However, in the presence of dominant negative inhibitors of CREB and c-Fos, these PTHrP effects were suppressed, and chondrocyte maturation was accelerated. Moreover, in combination, the effects of dominant negative c-Fos and CREB are synergistic, suggesting interaction between these signaling pathways during chondrocyte differentiation.

Identification of a Prg4-expressing articular cartilage progenitor cell population in mice.

To generate knockin mice that express a tamoxifen‐inducible Cre recombinase from the Prg4 locus (Prg4GFPCreERt2 mice) and to use these animals to fate‐map the progeny of Prg4‐positive articular cartilage cells at various ages.

Smad6 is induced by BMP-2 and modulates chondrocyte differentiation.

BMPs regulate cartilage differentiation and have been approved for clinical use as stimulators of bone repair. BMP signaling is complex and there are multiple potential points of regulation, including modulation of Smad signaling, which is inhibited by both Smad6 and Smad7. In the current manuscript we assessed the expression and biological function of Smad6 during chondrocyte differentiation. We found that the induction of chondrocyte differentiation by BMP‐2 in chicken sternal embryonic chondrocytes was accompanied by a marked increase in Smad6 mRNA and protein levels. A morpholino antisense oligonucleotide complementary to Smad6 reduced the expression of Smad6 protein and enhanced the stimulatory effect of BMP‐2 on both colX and alkaline phosphatase activity. In contrast, over‐expression of Smad6 blocked BMP‐2 mediated induction of the type X collagen promoter, b2‐640 Luc. Therefore, expression studies as well as gain and loss of function experiments suggest that Smad6 participates in an important negative feedback loop whereby BMP‐2 mediated effects on chondrocyte differentiation are reduced by induction of Smad6. Additional studies are required to determine the extent to which this pathway participates in pathologic processes involving cartilage.

CREB Cooperates with BMP-stimulated Smad signaling to enhance transcription of the Smad6 promoter

Growth plate chondrocytes integrate a multitude of growth factor signals during maturation. PTHrP inhibits maturation through stimulation of PKA/CREB signaling while the bone morphogenetic proteins (BMPs) stimulate maturation through Smad mediated signaling. In this manuscript, we show that interactions between CREB and the BMP associated Smads are promoter specific, and demonstrate for the first time the requirement of CREB signaling for Smad mediated activation of a BMP responsive region of the Smad6 promoter. The 28 base pairs (bp) BMP responsive element of the Smad6 promoter contains an 11 bp Smad binding region and an adjacent 17 bp region in which we characterize a putative CRE site. PKA/CREB gain of function enhanced BMP stimulation of this reporter, while loss of CREB function diminished transcriptional activity. In contrast, ATF‐2 and AP‐1 transcription factors had minimal effects. Electrophoretic mobility shift assay (EMSA) confirmed CREB binding to the Smad6 promoter element. Mutations eliminating binding resulted in loss of transcriptional activity, while mutations that maintained CREB binding had continued reporter activation by CREB and BMP‐2. The Smad6 gene was similarly regulated by CREB. Dominant negative CREB reduced BMP‐2 stimulated Smad6 gene transcription by 50%, but markedly increased BMP‐2 mediated stimulation of colX and Ihh expression. In contrast, PTHrP which activates CREB signaling, blocked the stimulatory effect of BMP‐2 on colX and Ihh, but minimally inhibited the stimulatory effect of BMP on Smad6. These findings are the first to demonstrate a cooperative association between CREB and BMP regulated Smads in cells from vertebrates and demonstrate that promoter‐specific rather than generalized interactions between PKA/CREB and BMP signaling regulate gene expression in chondrocytes. J. Cell. Physiol. 198: 428–440, 2004© 2003 Wiley‐Liss, Inc.

ATF-2 cooperates with Smad3 to mediate TGF-β effects on chondrocyte maturation

This study demonstrates that ATF-2 cooperates with Smad3 to regulate the rate of chondrocyte maturation in response to TGF-β. ATF-2 was rapidly phosphorylated in chick embryonic cephalic sternal chondrocytes following treatment with TGF-β, and the effect was dependent upon p38 kinase activity. Transient transfection of both wild-type ATF-2 or Smad3 activated the TGF-β-responsive reporter, p3TP-Lux, and synergistic effects were observed with ATF-2 and Smad3 coexpression. The effect of Smad3 and ATF-2 alone and in combination on chondrocyte maturation was examined in cultures simultaneously infected with RCAS viruses expressing different viral envelope proteins. When expressed alone, wild-type ATF-2 or Smad3 both inhibit colX expression and partially mimic the effects of exogenous TGF-β. However, in combination the effects were additive and similar to the inhibitory effects of TGF-β on colX expression. Loss of function experiments using dominant negative ATF-2 or Smad3 partially blocked the inhibitory effect of TGF-β on colX, while together the blockade was complete. Similar effects were observed with another TGF-β-responsive gene, PTHrP. However, the induction of colX by BMP-2 was not affected by overexpression of either wild-type or dominant negative ATF-2, indicating specificity for TGF-β signaling. In contrast, although TGF-β does not activate CRE/CREB signaling, dominant negative CREB enhanced colX expression in control and in TGF-β and BMP-2-treated cultures. Thus, ATF-2 regulates chondrocyte maturation as a direct target of TGF-β signaling while CREB regulates differentiation by targeting genes independent of the individual signaling effects of TGF-β or BMP-2.

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

During endochondral ossification, small, immature chondrocytes enlarge to form hypertrophic chondrocytes, which express collagen X. In this work, we demonstrate that 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. Our findings indicate that FoxA family members are crucial regulators of the hypertrophic chondrocyte differentiation program.

PTHrP expression in chick sternal chondrocytes is regulated by TGF-β through Smad-mediated signaling

PTHrP regulates the rate of chondrocyte differentiation during endochondral bone formation. The expression of PTHrP and its regulation by TGF‐β, BMP‐2, and PTHrP was examined in upper sternal chondrocytes following 1, 3, and 5 days of continuous treatment. While TGF‐β stimulated the expression of PTHrP (5‐fold), PTHrP caused a slight inhibition, and BMP‐2 markedly inhibited PTHrP mRNA expression. The effect of these factors on PTHrP expression was not simply related to the maturational state of the cells, since BMP‐2 increased, while both PTHrP and TGF‐β decreased the expression of type X collagen. TGF‐β isoforms 1, 2, and 3 all stimulated PTHrP expression. Signaling events involved in the induction of PTHrP by TGF‐β were further evaluated in a PTHrP‐promoter CAT construct. The effect of TGF‐β, BMP‐2, and PTHrP on the PTHrP‐promoter paralleled their effects on mRNA expression, with TGF‐β significantly increasing CAT activity, BMP‐2 decreasing CAT activity, and PTHrP having a minimal effect. Co‐transfection of the TGF‐β signaling molecule, Smad 3, mimicked the effect of TGF‐β (induction of PTHrP promoter), while dominant negative Smad 3 inhibited the induction of the PTHrP promoter by TGF‐β. Furthermore, infection with a Smad 3‐expressing retrovirus mimicked the effects of exogenously added TGF‐β, and induced PTHrP mRNA expression in the infected chondrocyte culture. In contrast, a dominant negative Smad 3 completely inhibited PTHrP promoter stimulation by TGF‐β, but only partially blocked the effect of TGF‐β on PTHrP mRNA synthesis. These findings demonstrate that PTHrP is expressed in chondrocytes undergoing endochondral ossification, and show regulation, at least in part, by TGF‐β through Smad mediated signaling events.

Prochondrogenic Signals Induce a Competence for Runx2 to Activate Hypertrophic Chondrocyte Gene Expression

Whereas Runx2 is necessary for bone formation and cartilage hypertrophy, it is unclear why Runx2 induces markers of chondrocyte hypertrophy only in chondrocytes. We document that chondrocytes either contain a cofactor, which can be induced in somitic cells by prochondrogenic signals, that is necessary for Runx2 to induce chondrocyte hypertrophy or, alternatively, lack a repressor of this maturation program. Sequential Shh and bone morphogenetic protein (BMP) signals or forced expression of either Nkx3.2 or Sox9 (plus BMP signals) induces chondrogenesis in presomitic mesoderm and simultaneously induces a competence for Runx2 to activate the chondrocyte maturation program. The ability of either sequential Shh and BMP signals or retrovirus‐encoded Nkx3.2 or Sox9 to induce this competence correlates with their ability to activate chondrogenesis in various embryonic tissues. Consistent with these findings in embryonic tissues, we have found that cotransfected Runx2 and Smad1 are able to induce the expression of a reporter construct driven by the collagen X regulatory sequences in chondrocytes but not in fibroblasts. In contrast, both Runx2 and Smad1 are competent to activate reporters driven by either reiterated Runx or Smad binding sites, respectively, in both cell types. As Sox9 and Nkx3.2 have previously been shown to block chondrocyte maturation in vivo, our findings suggest that these transcription factors can, in addition, either induce the expression or activity of a factor in chondrocytes that is required for Runx2 to activate the chondrocyte maturation program, or alternatively that these transcription factors block the expression or activity of a repressor of this maturation program. Developmental Dynamics 236:1954–1962, 2007.

BMP-mediated induction of GATA4/5/6 blocks somitic responsiveness to SHH

The relative timing of SHH and BMP signals controls whether presomitic mesoderm (PSM) cells will adopt either a chondrogenic or lateral plate mesoderm fate. Here we document that SHH-mediated induction of Nkx3.2 maintains the competence of somitic cells to initiate chondrogenesis in response to subsequent BMP signals by repressing BMP-dependent induction of GATA genes. Conversely, administration of BMP signals to PSM or forced expression of GATA family members in chick PSM explants blocks induction of hedgehog-dependent gene expression. We demonstrate that GATA factors can interact with Gli factors and can recruit the transcriptional co-factor FOG1 (ZFPM1) to the regulatory region of the mouse Gli1 gene, repressing the induction of Gli1 by SHH by binding to both GATA and Gli binding sites. Knockdown of FOG1 reverses the ability of GATA factors to repress Gli1 expression. Our findings uncover a novel role for GATA transcription factors as repressors of hedgehog signaling, and document that NKX3.2 maintains the ability of sclerotomal cells to express SHH transcriptional targets in the presence of BMP signals by repressing the induction of Gata4/5/6.

Identification of a Prg4-positive articular cartilage progenitor cell population

To generate knockin mice that express a tamoxifen‐inducible Cre recombinase from the Prg4 locus (Prg4GFPCreERt2 mice) and to use these animals to fate‐map the progeny of Prg4‐positive articular cartilage cells at various ages.