REGULATION - Primer on the Metabolic Bone Diseases and Disorders of

Transcriptional control of osteoblast differentiation

Several transcription factors (TF) are essential for the differentiation of MSCs into osteoblasts. Together they control expression of lineage‐specific genes though sequence‐directed binding to DNA. Although numerous transcription factors have been implicated in osteogenesis, this section will focus on TFs that have been studied in the most detail over the years.

Many of the pro‐osteogenic signaling pathways rely on the activity of Runx2 (Cbfa1/Aml3/Pebp2αA), which is considered the master regulator of osteoblast differentiation because Runx2 null mutations prevent bone formation in vivo. Specifically, Runx2 deletion prevents the differentiation of osteoblasts from MSCs and suppresses both intramembranous and endochondral bone development and mineralization. Elevated expression of Runx2 increases osteoclastogenesis [23], presumably by raising expression of RANKL in osteoblasts. Runx2 haplo‐

insufficiency causes cleidocranial dysplasia, which is characterized by the absence of clavicles and incomplete

development of cranial bones [24]. The interaction of Runx2 with coactivators and corepressors can either enhance or suppress the expression osteoblastic genes to ensure proper osteoblast differentiation [25].

The second crucial osteoblast‐specific TF is Sp7 (Osterix), which is downstream of Runx2. As with Runx2, deletion of the mouse Sp7 gene prevents osteoblast differentiation and bone formation [26]. Functionally, Sp7 interacts with NFATc1 and a variety of transcriptional cofactors to stimulate the expression of genes required for osteoblast differentiation [27].

A third key TF that contributes to osteoblast differentiation is ATF4, which forms a complex with Runx2 to stimulate the expression of osteogenic genes [28]. Lack of ATF4 delays bone formation and causes low bone mass.

Disruption of ATF4 transcriptional activity is linked to Coffin–Lowry syndrome, which is characterized by men- tal retardation and skeletal abnormalities [29]. Beyond Runx2, Sp7/Osterix, and ATF4, there are many other TFs that contribute to osteoblast differentiation, including Dlx3, Dlx5, Msx2, Tcf7/Lef1, Satb2, as well as steroid hormone receptors such as vitamin D receptor (Vdr), glucorti- coid receptor (GR/NR3C1), and estrogen receptor‐α (Erα/ ESR1). The combined biological activities of these TFs permit coordinate commitment of MSCs into the osteogenic lineage, but also control the differentiation process and the termination of the bone‐forming program.

Epigenetic transcriptional mechanisms controlling osteoblast differentiation

Transcriptional regulation of gene expression in osteoblasts is controlled by epigenetic events that are important for normal osteoblast differentiation and function.

Two major epigenetic mechanisms that control gene expression and function of osteoblasts are transcription‐

related chromatin modifications (DNA methylation and histone posttranslational modifications). These chemical changes to DNA and histones modulate the accessibility of TFs and RNA polymerase II to DNA. Additional posttranscriptional mechanisms (microRNAs and long‐noncoding RNAs) control mRNA stability and translation and are considered epigenetic.

DNA methylation at the fifth carbon of cytosine promotes gene silencing. This modification is actively regulated in osteoblasts and changes during differentiation and in response to external stimuli. Differentiation of MSCs into osteoblasts is accompanied by progres- sive DNA methylation of stem cell genes [30].

Hypomethylation in promoters of osteogenic genes occurs as precursor cells differentiate into osteoblasts.

For example, reduction in CpG methylation of the osteocalcin promoter enhances its expression [31].

Active DNA demethylation promotes osteogenic differentiation in vitro and in vivo [32]. Thus, the silencing of stem cell genes by DNA hypermethylation and the activation of osteoblast‐specific genes by DNA

Conclusion 35 hypomethylation is a fundamental epigenetic mecha-

nism that mediates osteogenic differentiation.

Core histone proteins (H2A, H2B, H3, and H4) undergo posttranslational modifications at several chemically labile amino acids (eg, lysine, arginine, and serine). Some histone modifications are associated with gene activation (eg, trimethylation at H3 lysine 4 [H3K4me3] or acetylation at lysine 27 [H3K27ac]), whereas others are more generally linked to gene silencing (eg, trimethylation at H3 lysine 27 [H3K27me3]). These and a plethora of other histone modifications collectively control gene expression. Changes in this “histone code” can modulate the osteogenic commitment of progenitor cells by modulating the accessibility of transcription factors such as Runx2 [33].

Acetylation of histones by acetyltransferases (eg, p300 and WDR5) is generally associated with gene activation during osteogenesis and these enzymes play crucial roles in osteogenic gene activation [34]. Removal of acetyl groups by histone deacetylases (HDACs) also contributes to osteoblastogenesis [35]. While enhancing global acetylation of histones promotes osteoblasts differentiation in vitro [36], alterations in these epigenetic marks nega- tively affect osteoblasts in vivo by reducing the number of osteoprogenitor cells [37].

Methylation of histones is also important in the differentiation and function of osteoblasts. Methylation of H3K27 promotes adipogenic differentiation, whereas demethylation promotes osteogenic differentiation of progenitor cells [38, 39]. Pro‐osteogenic effects of inacti- vating the H3K27 methyltransferase Ezh2 are evident in vivo [40]. Furthermore, genetic mutation of the H3K36 methyltransferase WHSC1, which is linked to Wolf–

Hirshhorn syndrome, causes craniofacial defects and growth abnormalities [41]. Consistent with this result, deletion of the NO66 gene, a histone H3K4/H3K36 demethylase, enhances bone formation by enhancing the expression of several osteogenic markers, including SP7 [42]. Thus, histone modifications play a critical role in osteoblast differentiation.

Epigenetic posttranscriptional mechanisms controlling osteoblast differentiation

Posttranslational control of gene expression during bone cell growth and differentiation is mediated by microR- NAs (miRNAs). These small noncoding RNAs (<24 nucleotides long) are produced by the RNA processing enzyme Dicer and bind to specific sequences in the 3′ untranslated regions (3′ UTRs) of their targets to control mRNA translation and/or degradation.

There is strong genetic and mechanistic evidence that miRNA function is important for normal skeletal development and osteoblast function in vivo. Genetic loss of Dicer during postnatal bone maturation results in a prominent increase in cortical bone volume, which is thought to be due to increased collagen expression [43]. There is clear evidence that miRNAs in general control osteoblast activity in cell culture, albeit few studies have specifically

examined miRNA effects in vivo. Two representative studies showed that some miRNAs block either alternative cell lineages or inhibitors that suppress osteogenesis. For example, the myogenic miR‐133 suppressed osteogenic commitment and the depletion of these molecules enhances osteogenic differentiation [44]. Conversely, up‐

regulation of miR‐218 suppressed inhibitors of WNT and BMP signaling to stimulate osteogenesis [45].

The different functions of miRNAs in skeletal development and disease have been well‐documented [46]. Beyond osteoblasts, these RNAs have key roles in the growth, differentiation, and function of mesenchymal stem cells, osteoclasts, chondrocytes, adipocytes, and myoblasts. One important theme that emerged from studies on miRNAs in osteoblasts is that they act through a “double‐negative principle” (eg, inhibiting repressors) to stimulate principal osteogenic signaling pathways (eg, Wnt signaling).

Similar to miRNAs, long noncoding RNAs (lncRNAs) are RNA transcripts longer than 200 nucleotides that do not encode proteins. While lncRNAs were initially considered random nonspecific transcripts, they are now known to play an essential role in controlling nuclear structure, gene expression, and cell differentiation [47].

While there are only a few studies that have assessed the role of lncRNAs in osteogenic differentiation, at least two lncRNAs (ANCR and HoxA‐AS3) alter the function of Ezh2 and heterochromatization in osteoblast progeni- tors through direct interaction [48, 49]. Disruption of the complex that these lncRNAs form with Ezh2 enhances osteogenic differentiation, similar to the finding that Ezh2 inhibition itself promotes bone formation in vitro and in vivo [39, 40]. LncRNA‐H19 has been shown to be pro‐osteogenic in MSCs by suppressing mRNA and protein expression of TGF‐β1 [50]. The mechanism of action of this lncRNA is complex. It involves the expression of miR‐675, which is expressed within an exon of lncRNA‐

H19, and also alters the expression of HDAC4 and HDAC5. As such, the proposed lncRNAs/miR‐675/

HDAC4/HDAC5 axis represents an intricate and pleio- tropic pathway that attenuates TGF‐β1 signaling and induces osteogenic commitment of MSCs.

CONCLUSION

Osteoblasts are mesenchymally‐derived cells that produce many factors essential for mineralization of the extracel- lular matrix. Osteoblasts coordinate bone remodeling and homeostasis with osteoclasts through paracrine signaling events. Within osteoblast nuclei, numerous transcription factors drive expression of tissue‐specific genes in a temporal and highly regulated fashion. Epigenetic events coordinated by histone or DNA‐modifying proteins and noncoding RNAs amplify and promote retention of tissue‐

specific functions. Understanding these events is essential for understanding bone degeneration and regeneration, and permits design of strategies that may prevent or mitigate bone‐related disorders.

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Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, Ninth Edition. Edited by John P. Bilezikian.

Companion website: www.wiley.com/go/asbmrprimer

5 INTRODUCTION

In the adult skeleton, osteocytes make up over 90–95%

of all bone cells compared with 4–6% osteoblasts and around 1–2% osteoclasts. These cells are regularly dis

persed throughout the mineralized matrix, connected to each other and cells on the bone surface through den

dritic processes generally radiating towards the bone surface and the blood supply. The dendritic processes travel through the bone in tiny canals called canaliculi (250–300 nm) whereas the cell body is encased in a lacuna (15–20 μm) (see Figs 5.1, 5.2, and 5.3). Osteocytes are thought to function as a network of sensory cells mediating the effects of mechanical loading through this extensive lacunocanalicular network. Not only do these cells communicate with each other and with cells on the bone surface, but their dendritic processes can extend past the bone surface into the bone marrow and vascular spaces. Osteocytes have long been thought to respond to mechanical strain to send signals of resorp

tion or formation, and evidence is accumulating to show that this is a major function of these cells. The number of functions attributed to these cells has been expanding [1] and includes regulation of phosphate homeostasis; therefore, the osteocyte network func

tions as an endocrine gland [2]. Defective osteocyte function may play a role in a number of bone diseases, especially glucocorticoid‐induced bone fragility and osteoporosis in the adult, and in nonbone disease such as chronic kidney disease and skeletal and cardiac muscle function [3].

Dalam dokumen Primer on the Metabolic Bone Diseases and Disorders of (Halaman 60-64)