MMP-13 is one of the critical mediators of the effect of HDAC4 deletion on the skeleton

doi:10.1016/j.bone.2016.06.010

Bone

Volume 90, September 2016, Pages 142-151

https://doi.org/10.1016/j.bone.2016.06.010 Get rights and content

Highlights

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We generated Hdac4-/-Mmp13^-/- knockout mice and determined if deletion of MMP-13 rescued the Hdac4^-/- mouse phenotype.
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Hdac4^-/- mice lacking Mmp13 exhibit partial recovery of the premature phenotype.
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MMP-13 in the Hdac4^-/- mice influences ALP, Mmp9 and TRAP expression.
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Increased MMP-13 in Hdac4^-/-- mice affects cortical bone area, architecture and trabecular bone mineral density and thickness.

Abstract

Histone deacetylase 4 (Hdac4) regulates chondrocyte hypertrophy. Hdac4^−/− mice are runted in size and do not survive to weaning. This phenotype is primarily due to the acceleration of onset of chondrocyte hypertrophy and, as a consequence, inappropriate endochondral mineralization. Previously, we reported that Hdac4 is a repressor of matrix metalloproteinase-13 (Mmp13) transcription, and the absence of Hdac4 leads to increased expression of MMP-13 both in vitro (osteoblastic cells) and in vivo (hypertrophic chondrocytes and trabecular osteoblasts). MMP-13 is thought to be involved in endochondral ossification and bone remodeling. To identify whether the phenotype of Hdac4^−/− mice is due to up-regulation of MMP-13, we generated Hdac4/Mmp13 double knockout mice and determined the ability of deletion of MMP-13 to rescue the Hdac4^−/− mouse phenotype. Mmp13^−/− mice have normal body size. Hdac4^−/−/Mmp13^−/− double knockout mice are significantly heavier and larger than Hdac4^−/− mice, they survive longer, and they recover the thickness of their growth plate zones. In Hdac4^−/−/Mmp13^−/− double knockout mice, alkaline phosphatase (ALP) expression and TRAP-positive osteoclasts were restored (together with an increase in Mmp9 expression) but osteocalcin (OCN) was not. Micro-CT analysis of the tibiae revealed that Hdac4^−/− mice have significantly decreased cortical bone area compared with the wild type mice. In addition, the bone architectural parameter, bone porosity, was significantly decreased in Hdac4^−/− mice. Hdac4^−/−/Mmp13^−/− double knockout mice recover these cortical parameters. Likewise, Hdac4^−/− mice exhibit significantly increased Tb.Th and bone mineral density (BMD) while the Hdac4^−/−/Mmp13^−/− mice significantly recovered these parameters toward normal for this age. Taken together, our findings indicate that the phenotype seen in the Hdac4^−/− mice is partially derived from elevation in MMP-13 and may be due to a bone remodeling disorder caused by overexpression of this enzyme.

Introduction

Histone deacetylase 4 (Hdac4) is a member of the class IIa HDACs and plays a central role in the formation of the skeleton [1], [2]. Hdac4 is expressed in prehypertrophic chondrocytes, and mice with global deletion of HDAC4 are significantly smaller than wild type mice and die during the first week of life. This is mainly due to ectopic ossification of endochondral cartilage, which prevents expansion of the rib cage and leads to an inability to breathe [2]. The formation of cartilaginous templates of endochondral bones occurs normally in the Hdac4^−/− mice, but the onset of chondrocyte hypertrophy is accelerated and, consequently, endochondral mineralization occurs precociously, leading to ectopic bone formation [2].

Chondrocyte hypertrophy is the final step in chondrocyte differentiation, in which the cartilage extracellular matrix (ECM) becomes calcified and partially degraded. Ossification begins when hypertrophic chondrocytes undergo programmed cell death and the calcified cartilage is invaded by blood vessels, osteoblasts, osteoclasts and mesenchymal precursors and forms primary ossification centers. Within these centers, the hypertrophic cartilage matrix is degraded, the hypertrophic chondrocytes die, and bone replaces the disappearing cartilage. Studies have shown that chondrocyte apoptosis does not lead to endochondral ossification [3]. Angiogenesis has been implicated as a crucial step in endochondral ossification [4], [5], [6], and degradation and remodeling of the cartilage matrix is essential for vascular invasion.

Matrix metalloproteinase 13 (Mmp13, also called collagenase-3) plays an important role in the degradation of components of the cartilage ECM. MMP-13 is expressed in hypertrophic chondrocytes and osteoblasts and promotes the removal of hypertrophic cartilage from the growth plate and the remodeling of newly deposited trabecular bone during long bone development [7], [8]. It degrades collagen type II efficiently but also collagen types I, III, and X, which are the major components of cartilage and bone [9]. It has been shown that MMP-13 acts directly during the initial stages of cartilage ECM degradation (the rate-limiting process for chondrocyte programmed cell death) and during angiogenesis prior to invasion by blood vessels and osteoclasts [8]. A mutation in the human Mmp-13 gene causes the Missouri variant of spondyloepimetaphyseal dysplasia (SEMD), a syndrome with abnormalities in development and growth of endochondral skeletal elements [10]. Mmp-13 null mice show no overt phenotypic abnormalities, they are fertile and have a normal lifespan but show abnormal growth plates and delayed ossification [7], [8]. Clinical investigation has shown that patients with articular cartilage destruction have high Mmp-13 expression [11], [12], suggesting that increased Mmp-13 may be associated with cartilage degradation. Studies have also shown that Mmp-13-overexpressing transgenic mice develop a spontaneous osteoarthritis (OA)-like articular cartilage destruction phenotype [5].

Recently, our laboratory showed that Hdac4 represses Mmp13 expression through inhibiting the activity of Runx2, and that Hdac4^−/− mice displayed increased Mmp-13 mRNA and protein levels in hypertrophic chondrocytes and trabecular bone [13]. In the present study, we generated double knockout mice (Hdac4^−/−/Mmp13^−/−) to determine whether the elevation of MMP-13 contributes to the phenotype of Hdac4^−/− mice.

Section snippets

Mice

Breeding pairs of heterozygous Hdac4 mice (Hdac4^+/−) on a C57BL/6 background were generated as described [1]. These heterozygous mice have no phenotypic abnormalities as described previously in [2]. Heterozygous or homozygous Mmp13-deficient mice (Mmp13^−/−) mice on a C57BL/6 background were a kind gift of Dr. Jeanine D'Armiento (Columbia University College of Physicians and Surgeons, New York). We characterized 5 and 8-day-old male and female mice, wild type, Hdac4^−/−, Mmp-13^−/− and Hdac4^−/−/

Mmp13 ablation in HDAC4^−/− mice increased their body weight, length and survival rate

To investigate the contribution of MMP-13 elevation to the premature phenotype seen in the Hdac4^−/− mice, we generated double knockout mice (Hdac4^−/−/Mmp13^−/−). Hdac4^−/− mice were severely runted (Fig. 1A, B) and died during the first week of life (Fig. 1C). Mmp13^−/− mice showed no gross phenotypic abnormalities (Fig. 1A), were fertile and had a normal lifespan. The Hdac4^−/−/Mmp13^−/− double knockout mice were bigger, heavier, and survived longer when compared with Hdac4^−/− mice (Fig. 1). These

Discussion

Our initial goal was to determine whether elevations in MMP-13 in Hdac4^−/− mice contribute to their skeletal phenotype. To that end, we generated Hdac4^−/−/Mmp13^−/− double knockout mice. Hdac4^−/− mice display dwarfism and do not survive to weaning. In the present paper, we show that Hdac4^−/− mice lacking Mmp13 have improved body weight, length and survival. In our previous study we showed that Hdac4 ablation in mice resulted in increased Mmp-13 expression and protein levels in vivo. Heterozygous

Acknowledgements

We thank Dr. Eric Olson and Dr. Jeanine D'Armiento for kindly giving us heterozygous Hdac4 deficient mice (Hdac4^+/−), and heterozygous or homozygous Mmp13-deficient mice (Mmp13^−/−) mice on a C57BL/6 background, respectively. We thank Dr. Malvin Janal for advice on statistical analyses. This work was funded by an NIH grant to N.C.P. (DK47420). The microCT was funded by NIH grant OD010751 (to N.C.P.).

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This chapter focuses on the hormone produced by the parathyroid gland, parathyroid hormone (PTH), its structure, regulation, action through its known receptors, physiologic functions, and diseases. The parathyroid glands were discovered in the late 19th century and were found to be distinct from the thyroid glands and to regulate Ca²⁺ metabolism. The hormone was purified in 1959 and found to be an 84 amino acid peptide, which was later synthesized. Its biologic activity is contained within the first 34 amino acids. Release of PTH from the gland is regulated by extracellular Ca²⁺ through the calcium-sensing receptor (high inhibits, low stimulates), by phosphate (high stimulates) and by 1,25-dihydroxyvitamin D3 (negative feedback inhibition). In this manner, the hormone regulates the levels or synthesis of these three components, by action primarily on bone and kidney. It acts through its receptor, PTHR1 (a G protein–coupled receptor), in the kidney and on cells of the osteoblast lineage in bone. PTH induces the synthesis of 1,25-dihydroxyvitamin D3 by the kidney, which acts on the intestine to increase uptake of Ca²⁺. PTH-PTHR1 binding facilitates increased renal reabsorption of Ca² and causes phosphaturia by downregulation of sodium/phosphate transporters. In bone, it causes many changes in gene expression through a number of signal transduction pathways in cells of the osteoblastic lineage, resulting in either catabolic or anabolic effects on bone. Because of the latter effects, PTH (1–34; teriparatide) was approved for treatment of osteoporosis. There are many diseases associated with each of these constituent parts, and these are described in the chapter. There are still gaps in our knowledge, and we have attempted to point these out.
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This chapter focuses on the hormone produced by the parathyroid gland, parathyroid hormone (PTH), its structure, regulation, action through its known receptors, physiologic functions, and diseases. The parathyroid glands were discovered in the late 19th century and were found to be distinct from the thyroid glands and to regulate Ca²⁺ metabolism. The hormone was purified in 1959 and found to be an 84 amino acid peptide, which was later synthesized. Its biologic activity is contained within the first 34 amino acids. Release of PTH from the gland is regulated by extracellular Ca²⁺ through the calcium-sensing receptor (high inhibits, low stimulates), by phosphate (high stimulates) and by 1,25-dihydroxyvitamin D3 (negative feedback inhibition). In this manner, the hormone regulates the levels or synthesis of these three components, by action primarily on bone and kidney. It acts through its receptor, PTHR1 (a G protein–coupled receptor), in the kidney and on cells of the osteoblast lineage in bone. PTH induces the synthesis of 1,25-dihydroxyvitamin D3 by the kidney, which acts on the intestine to increase uptake of Ca²⁺. PTH-PTHR1 binding facilitates increased renal reabsorption of Ca² and causes phosphaturia by downregulation of sodium/phosphate transporters. In bone, it causes many changes in gene expression through a number of signal transduction pathways in cells of the osteoblastic lineage, resulting in either catabolic or anabolic effects on bone. Because of the latter effects, PTH (1–34; teriparatide) was approved for treatment of osteoporosis. There are many diseases associated with each of these constituent parts, and these are described in the chapter. There are still gaps in our knowledge, and we have attempted to point these out.
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Full Length ArticleMMP-13 is one of the critical mediators of the effect of HDAC4 deletion on the skeleton

Highlights

Abstract

Introduction

Section snippets

Mice

Mmp13 ablation in HDAC4−/− mice increased their body weight, length and survival rate

Discussion

Acknowledgements

Cell

Mech. Dev.

Mech. Dev.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Osteoarthr. Cartil.

Osteoarthr. Cartil.

Bone (NY)

Bone (NY)

Mol. Cell Biol. Res. Commun.

Dev. Cell

The many roles of histone deacetylases in development and physiology: implications for disease and therapy

Nat. Rev. Genet.

VEGFA is necessary for chondrocyte survival during bone development

Development and Disease

Critical roles for collagenase-3 (Mmp13) in development of growth plate cartilage and in endochondral ossification

Proc. Natl. Acad. Sci. U. S. A.

Altered endochondral bone development in matrix metalloproteinase 13-deficient mice

Development

Full Length Article
MMP-13 is one of the critical mediators of the effect of HDAC4 deletion on the skeleton

Mmp13 ablation in HDAC4^−/− mice increased their body weight, length and survival rate