Molecular mechanisms regulating osteoblast differentiation: miR-322/Tob2 and PI3K/SMAD networks

Abstract

We propose a molecular description of the mechanism whereby the osteogenic master gene Osx is controlled post-transcriptionally through a mechanism driven by miR-322/Tob2, strongly suggesting that control of specific mRNA decay is relevant in bone development and homeostasis. miR-322 has been previously studied together with miR-503 in myogenesis as promoters of cell cycle quiescence and differentiation by down-regulation of Cdc25A. Our results also show that, after differentiation by BM P-2, the miR-322 level progressively decreases in C2C12 and MC3T3-E1 cells and primary cultures of BM-MSCs. We have mentioned the up-regulated expression of osteogenic transcription factors during BMP-2 treatment. Then, miR-322, by means of Tob2 down-regulation, adjusts the expression levels of some of these factors, particularly Osx. This profile seems to allow the transcriptional up-regulation of the osteogenic transcription factors, whereas miR-322 may later exert a regulatory mechanism that allows fine-tuning of bone homeostasis.

Tobi and Tob2 proteins constitute a Tob subfamily and belong to the BTG/Tob antiproliferative factor protein family. Tob genes have emerged as key players in mediating post-transcriptional gene expression by regulating mRNA deadenylation and therefore cytoplasmic mRNA levels. Our results suggest that new target genes displaying compatible Tob-interacting secondary structures at their 3'-UTR could be subjected to specific mRNA repression by Tob family members, as we suggest here for Osx. These data are in agreement with previous research showing that, although Tobi knock-out mice are born without apparent phenotypic abnormalities, Tobi-deficient adult mice were shown to have higher bone mass compared with wild-type mice.

It has been shown that Tob proteins can interact with CPEB2-4 and specifically intensify the rapid decay of particular transcripts. Their RNA-binding domains recognize mRNAs with specific secondary structures containing U-rich loops and interact with single-stranded uridines as well as double-stranded stems present in the 3'-UTR of the target mRNA. Recent studies showed Tobi interaction with cytoplasmic CPEB2-4, which negatively regulate the expression of a target by tethering to specific mRNA and mediating recruitment of the deadenylase Cnot7, leading to specific mRNA decay. The Osx 3'-UTR contains secondary structures with a stem-loop structure similar to those bound by CPEB2-4. Our RNA pulldown analysis showed that these sequences are directly bound by CPEB proteins and Tob2. Thus, in view of our results, we hypothesize that Osx mRNA could be bound by Tob2 and CPEB2-4 and, as a consequence, specifically degraded. Future work is necessary to discern which CPEB-like proteins are involved in the interaction between Tob2 and stem-loop structures in the 3'-UTR of Osx and other osteogenic genes.

2. Class I PI-3-Kinase signaling is critical for bone formation through regulation of SMADi activity in osteoblasts

The present work reveals that PI3Ka and (3 isoforms are major regulators of osteoblast differentiation and survival. Deletion of either p110alpha and/or piio6 impairs osteoblast differentiation by decreasing the expression of key osteoblast-determining transcription factors and their transcriptional targets. More importantly, these results establish a network of molecular events that integrate distinct osteogenic inputs such as IGFi, Wnts and BMPs. Signals from these cytokines converge on GSK3 inhibition and higher SMADi levels, which confers a larger response to the osteogenic BMP action on osteoblasts.

Our results confirm that p110alpha is critical for early bone formation and development and further demonstrate this requirement for postnatal bone homeostasis. Deletion of p110alpha during early bone development causes an osteopenic phenotype in bones of both endochondral and intramembranous origin. We also took advantage of an inducible Cre system to delete p110alpha at 1-2 days of age, when bone architecture is already established and BMD and cortical thickness were approximately 4o-5o% of that of adult mice. Postnatal p110alpha deletion also led to a significant loss of either calvarial, cortical or trabecular bone at 12 weeks of age.

When p110alpha was deleted, although a slow proliferation rate and higher sensitivity to apoptosis was seen in cultured osteoblasts in vitro, there were no significant changes in the total number and proliferation rate of osteoblasts in bones of prim-deficient mice. These data mirror those previously obtained in Pten-deficient mice. Moreover, deletion of p1106 did not increase sensitivity to apoptosis in osteoblasts, but it also produced a strong bone phenotype. In our mouse model, lack of PI3K isoforms leads to similar changes in the expression of osteoblast-specific transcription factors in calvaria, long bone and osteoblast cultures. Whereas Runx2 expression was significantly reduced only after deletion of both p110alpha and piio6, Osx expression was strongly suppressed in all cases. OSX has been shown to transcriptionally regulate the expression of Cohal, Ibsp, Bglap and Fmod. Thus, lower levels of OSX could account for impaired osteoblast maturation and function, through decreased transcription of these genes..

GSK3 is a multifunctional kinase that is constitutively active and negatively regulated by numerous signaling pathways such as PI3K/AKT and canonical Wnt. Evidence suggests a negative role for GSK3 activity in osteogenesis. Our data identified GSK3 activity as a novel node of integration for multiple osteogenic signals. Previous studies have shown that MAPK and GSK3 pathways can interfere with BMP signaling. SMADi is sequentially phosphorylated on its linker region by MAPKs and GSK3. The latter modification primes for the recognition and polyubiquitination of SMAD1 by the SMURF1 and -2 E3 ubiquitin ligases. Thus, GSK3-regulated cellular levels of SMAD1 integrate signals from PI3K activators and Wnts with those of the BMPs to give a coordinated osteogenic readout. Our results conclude that genetic or pharmacological inhibition of PI3K blocks the inhibitory phosphorylation of GSK3 and regulates SMAD1 protein stability. These effects on SMADi levels were partially reversed by pharmacological inhibition of GSK3. The effects on osteoblast-specific gene expression could be reversed by ectopic expression of exogenous SMAD1. Further cooperation comes from the fact that GSK3 activity also governs nuclear levels of (3-catenin. Moreover, SMAD1 and 13-catenin transcriptionally cooperate in key osteogenic gene promoters. Thus, the present findings represent a molecular framework to understand the mounting evidence showing cooperative activation of osteoblast differentiation and function by IGFs, Wnts and BMPs.

En esta tesis se han estudiado y demostrado distintos mecanismos moleculares involucrados en la diferenciación osea.

Los microRNAs emergieron hace pocos años como reguladores post-transcripcionales presentes en todos los procesos biológicos. En esta tesis se describe el microRNA-322 como un miRNA reprimido durante el tratamiento con BMP-2 en células osteoblásticas y se detalla cómo éste actúa sobre el extremo 3'-UTR del mRNA de Tob2, produciendo su degradación. TOB2 es una proteína que forma parte de la familia de proteínas antiproliferativas TOB2/BTG. Tob2 se ha descrito como mediador de modificaciones post-traduccionales regulando la deadenilación general de los mRNAs. A demás, se ha visto que participa acelerando específicamente la degradación de determinados mRNAs al unirse a proteínas CPEBlike (CPEB2-4). En la presente tesis se describe el mecanismo por el cual el miR-322 degrada específicamente Tob2 y lo retiene de ejercer su función específica sobre Osterix. El uso de inhibidores específicos y los estudios con ratones mutantes existentes hasta el momento sugerían un papel importante para la proteína PI3-quinasa en el desarrollo y mantenimiento óseo. En esta tesis se describen los fenotipos derivados de la deleción específica en osteoblastos de las isoformas más abundantes en hueso (p110alpha, p 1 10beta y p 1 10alpha/beta). La ausencia de cualquiera de las subunidades catalíticas de PI3K se traduce en una reducción de masa ósea cuando los fémures de dichos ratones son analizados por microtomografia computerizada (micro-CT). El estudio in vitro de osteoblastos primarios obtenidos de los mismos ratones muestran una disminución de los principales genes osteogénicos a nivel de mRNA y proteína, concluyendo que la deficiencia de PI3K en la célula provoca una menor diferenciación osteoblástica. PI3K regula, a través de la fosforilación de AKT, un gran número de efectores. Entre ellos está GSK3, quinasa que se fosforila e inactiva en respuesta a PI3K. Estudios anteriores a esta tesis describen cómo GSK3 fosforila la región "linker" de Smadl (proteína efectora de la vía de BMPs), marcándola para degradación. En concordancia con dichos trabajos, tanto los ratones como los osteoblastos primarios defectivos en PI3K presentan menor cantidad de SMAD1 y de SMAD1 fosforilado, constituyendo así una mecanismo en el cual la falta de PI3K, a través de una menor inactivación de GSK3, aumentaría la degradación de Smads. Dicho mecanismo describe un eje PI3K-GSK3-SMAD en el que la deficiencia de PI3K conlleva a un desarrollo óseo deficitario.