Elsevier

Bone

Volume 51, Issue 3, September 2012, Pages 407-417
Bone

Original Full Length Article
The intraflagellar transport protein IFT80 is required for cilia formation and osteogenesis

https://doi.org/10.1016/j.bone.2012.06.021Get rights and content

Abstract

Intraflagellar transport (IFT) proteins are essential for the assembly and maintenance of cilia, which play important roles in development and homeostasis. IFT80 is a newly defined IFT protein. Partial mutation of IFT80 in humans causes diseases such as Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III with abnormal skeletal development. However, the role and mechanism of IFT80 in osteogenesis is unknown. Here, we first detected IFT80 expression pattern and found that IFT80 was highly expressed in mouse long bone, skull, and during osteoblast differentiation. By using lentivirus-mediated RNA interference (RNAi) technology to silence IFT80 in murine mesenchymal progenitor cell line-C3H10T1/2 and bone marrow derived stromal cells, we found that silencing IFT80 led to either shortening or loss of cilia and the decrease of Arl13b expression — a small GTPase that is localized in cilia. Additionally, silencing IFT80 blocked the expression of osteoblast markers and significantly inhibited ALP activity and cell mineralization. We further found that IFT80 silencing inhibited the expression of Gli2, a critical transcriptional factor in the hedgehog signaling pathway. Overexpression of Gli2 rescued the deficiency of osteoblast differentiation from IFT80-silenced cells, and dramatically promoted osteoblast differentiation. Moreover, introduction of Smo agonist (SAG) promotes osteoblast differentiation, which was partially inhibited by IFT80 silencing. Thus, these results suggested that IFT80 plays an important role in osteogenesis through regulating Hedgehog/Gli signal pathways.

Highlights

► IFT80 was highly expressed in bone tissue and during osteoblast differentiation. ► Silencing IFT80 led to either shortening or loss of cilia and blocked osteogenesis. ► Overexpression of Gli2 rescued osteogenic deficiency of IFT80-silenced cells. ► Introduction of SAG promoted osteogenesis, which was inhibited by silencing IFT80. ► IFT80 stimulates osteogenesis through regulating Hedgehog/Gli signal pathway.

Introduction

Primary cilia are microtubule-based organelles localized on the surface of almost all vertebrate cells including osteoblasts and osteocytes. These organelles are extended and maintained by the transport of particles along the axoneme mediated by intraflagellar transport (IFT) bidirectional machinery. IFT proteins are organized into two complexes. Complex A containing IFT144, 140, 139, and 122 proteins mediates retrograde transport of cargoes from the tip to the base of the cilia, while complex B containing IFT172, 88, 81, 80, 74/72, 57/55, 52, 46, 27, and 20 mediates anterograde transport of specific cargoes from the base to the tip. The movement of IFT proteins is carried out by two different microtubule-based motors: the anterograde (towards the cilia tip) motor is kinesin-II, which is composed of Kif3a and Kif3b motor subunits; the retrograde (towards the cell body) motor is cytoplasmic dynein-Ib. IFT complexes carry axonemal subunits to the site of assembly at the tip of the axoneme and are necessary for axonemal growth [1]. In recent years, the importance of IFT proteins for the development and function of the skeleton has been demonstrated due to the findings of skeletal abnormalities in human cilia-associated disorders [2], [3], [4], [5], [6] and in IFT-related mouse knockout studies [7], [8], [9], [10], [11], [12], [13]. Increasing studies have shown that primary cilia/IFT regulate embryonic bone development [8], [14], [15], [16], [17], [18] and mechanically regulate bone formation in adults [19], [20]. However, the function and mechanism of IFT/cilia proteins in osteoblast differentiation and function are still largely undefined.

Most recently, Xiao et al. [21] reported that targeted deletion of PKD1 (polycystin-1) in osteoblasts results in osteopenia phenotype, and impaired osteoblastic differentiation. While Kif3a deficiency reverses the skeletal abnormalities in Pkd1 deficient mice by restoring the balance between osteogenesis and adipogenesis [18]. Qiu et al. [17] demonstrated that osteoblast specific deletion of Kif3a causes increased cell proliferation, impaired osteoblastic differentiation, and enhanced adipogenesis in vitro. They further found that conditionally deleted Kif3a in osteoblasts results in the reduction or shorten of primary cilia and develops osteopenia in vivo and suggested that Kif3a regulates osteoblastic differentiation and function through multiple pathways including hedgehog, intracellular calcium and Wnt signaling. These findings highlighted important roles of IFT and cilia related proteins in osteoblast differentiation and bone development.

A number of studies have shown that the skeletal phenotypes observed in a variety of IFT and ciliary component knockout lines can be attributed to abnormal hedgehog signaling (Hh) [8], [12], [22]. Hh signaling is one of the major signaling pathways that regulate osteogenesis and embryonic bone development and post-embryonic bone homeostasis [23], [24]. In vertebrates, the Hh family consists of three members: Sonic Hh (Shh), Indian Hh (Ihh), and Desert Hh (Dhh) [24]. Hh protein binding to the transporter-like receptor Patched (Ptch) releases Ptch inhibition of Smoothened (Smo) allowing the transduction of the Hh signal to the primary cilium. This in turn activates Gli transcription factors that mediate the transcription of Hh target genes in cells [25], [26], [27]. Without a cilium, hedgehog signaling is abrogated, leading to a variety of skeletal malformations as well as embryonic lethality. For example, deletion of IFT88 in limb mesenchyme resulted in shortening of the bone in the limbs due to alterations in Ihh signaling and endochondral bone formation [8]. Conditional deletion of IFT88 or Kif3α in chondrocyte lineage by using Col2α1-cre lead to abnormal hedgehog signaling topography and apparent growth plate dysfunction [22], [28], which are similar to conditional deletion of Ihh in postnatal cartilage (Ihhflox/flox, Col2a-CreER) [29].

IFT80 is a newly identified IFT protein, which encodes a 777-residue protein that contains seven WD40 domains and is a component of the IFT complex B [30]. WD40 domains are short motifs of approximately 40 amino acids that form circular beta propeller structures. During intraflagellar transport, this complex helps carry materials from the base to the tip of cilia. Partial mutations of IFT80 in humans cause Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly type III (SRPIII). Both diseases have severe bone abnormalities including shortening of the long bones and constriction of the thoracic cage [31], [32], [33]. SRP type III is a more severe disorder with a range of extra skeletal malformations, including cleft lip or palate, cystic renal disease, gastrointestinal, urogenital, brain and/or cardiac malformations. These two diseases often lead to death prenatally or in infancy due to respiratory insufficiency. However, currently, it is still unclear if the abnormal bone phenotype result from the effect of IFT80 mutation on osteogenesis or indirect effect of mutation of IFT80 in human tissues. Therefore, in this study, to identify the role and mechanism of IFT80 in osteoblast differentiation, we first identified the gene expression pattern of this newly discovered protein in various mouse tissues, including skull and bone among others, and confirmed IFT80 is predominantly expressed in bone as well as during osteoblast differentiation. We further determined the effect of IFT80 on osteoblast differentiation and activation and on the Hh/Gli signaling transduction pathway. Our results demonstrated that the IFT80 gene plays an essential role in osteoblast differentiation and likely is involved in Hh/Gli signal pathway.

Section snippets

Cell lines and cell culture

HEK293T human embryonic kidney cell line, C3H10T1/2 murine mesenchymal progenitor cell line and RAW264.7 murine monocyte/macrophage cell line were obtained from American Type Culture Collection (ATCC). For preparation of mouse BMMs and BMSCs, animal procedures were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of University at Buffalo (UB). Both femora and tibias were removed and soft tissues were detached from the bone of

Ift80 is highly expressed in osteoblast progenitor cells, osteoblasts, and bone

To identify the expression pattern of IFT80, we first analyzed IFT80 mRNA expression on 10 kinds of mouse tissues by performing real time RT-PCR. The result showed that IFT80 was highly expressed in the long bone and skull. It is abundant in eye, lung, spleen and kidney tissues. IFT80 was also expressed in muscle, heart, liver, and brain tissues, but to a far lesser extent (Fig. 1A). This result was further confirmed by immunostaining in mouse long bone, eye and kidney tissues (Fig. 1B). To

Discussion

It has been reported that partially mutation of IFT80 in human causes diseases such as Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III with abnormal skeletal development [31], [33], [63]. However, based on these mutation observations, it is still unclear if the abnormal skeletal phenotype were resulted from indirect effect of mutation of IFT80 in human tissues or due to the effect of IFT80 mutation on osteogenesis. Therefore, in this study, we have

Acknowledgments

We thank Drs. Rosemary Dziak, Douglas Olson and Mr. David Hadbawnik for critical reading of the manuscript, and Dr. Wade J. Sigurdson, the director of the Confocal Microscope Facility in the School of Medicine and Biomedical Sciences, University at Buffalo for the assistance with fluorescence microscopy. This work was supported by a National Institute of Health grant AR055678 (S. Yang) and AR061052 (S. Yang).

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