The present study is a genome-wide microarray analysis of bone lining cells from the normal developing rat bulla aiming to identify bone-related candidate genes and gene sets in the MEACS. FDR was used to assess significance: If a gene product value is highly variable even a large FC between, for example, young and adult IB might represent only chance variation, and not a true biological difference. However, if stochastic variation is small, even a slight difference may be significant. By analyzing statistically the internal variance of triple measurements the differentially expressed genes could be identified with a specified risk of error in case of larger as well as smaller FCs. The single gene analysis identified 22 differentially expressed genes related to bone metabolism when adult (quiescent) IB was compared with young (resorbing) IB (FDR < 0.05). Other genes may be biologically relevant if they work together as a functional group even if they fail to reach significance when analyzed separately: a differential group effect may become significant only when the corresponding group members are analyzed together as a gene set. Thus, a gene set analysis on public and novel gene sets was done. This identified five enriched bone-related gene sets (P < 0.05).
In the following paragraphs some proteins and gene sets of particular interest are discussed. They were selected on a fold change or relevance to previously published results.
Proteins Previously Highlighted in Publications on Middle Ear Bone Metabolism
Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases. Recently, a study based on oligonucleotide microarray analyses identified upregulation of MMP10 in cholesteatoma extracellular matrix using retroauricular skin as control (Kwon et al., 2006). MMP10 has also been documented in osteoclasts in osteophytic bone and neonatal ribs in a study using immunohistochemistry and in situ zymography (Bord et al., 1998). This study demonstrated an upregulation of MMP10 (FC-5.0). The MMP10 seems to play a role as a regulator of bone resorption in pathological and healthy MEACS and may potentially have a bone-signaling function.
Tenascins (TNs) are extracellular matrix glycoproteins. There are four members of the TN family: TNC, TNR, TNX, and TNN. Tenascins have previously been implicated in middle ear signaling, but the immunohistochemical tools used in the studies did not allow discrimination between the four members (Lang et al., 1997; Juhasz et al., 2009). In the present study, the TNN was upregulated in young IB (FC −5.0) and may have a possible bone-signaling role in the middle ear.
Novel Bone-Related Proteins of the Middle Ear
Calcium-binding protein molecule A8 (S100A8) and calcium-binding protein molecule A9 (S100A9) are involved in the regulation of a number of cellular processes. They are localized in the cytoplasm and/or nucleus of a wide range of cells including osteoblasts and osteoclasts (Zreiqat et al., 2007). Recently, S100A8 was shown to stimulate osteoclast differentiation and activity during space flight (Sambandam et al., 2010). In this study S100A8 (FC −25.0) and S100A9 (FC −16.7) were upregulated in young IB implicating a potential effect on the osteoblasts and osteoclasts in the normal growing MEACS.
Calpains (CAPNs) form a group of 14 members of calcium-sensitive cysteine proteases. In a biochemical study CAPN I and II were suggested to participate in the middle ear bone resorption associated with cholesteatoma by their involvement in collagen destruction (Amar et al., 1996). In addition, CAPN6 has been identified as a regulator of osteoclastic bone resorption via cytoskeletal organization and microtubule acetylation (Hong et al., 2011). In the present study a significant upregulation of CAPN6 (−16.7) in young IB was demonstrated.
Fibrillin-1 (FBN1) is an extracellular matrix glycoprotein that is altered in Marfan syndrome, characterized by, for example, defects in the skeletal system. Recently, FBN1 and FBN2 were found to be implicated in bone formation through a process involving the regulation of local TGFβ and bone morphogenetic protein signals (Nistala et al., 2010). In accordance with these results, this study showed an upregulation of FBN1 on young IB (FC −5.0). Therefore, FBN1 may be a medium range signaling protein for coupled bone formation on OB.
Calcitonin-related polypeptide (CALC) is a neuropeptide which exists in two forms, CALCA and CALCB. The MEACS might be under control of the nervous system as denervation resulted in bone resorption inside the bulla of the Mongolian gerbil (Sherman and Chole, 1996; Sherman and Chole, 1999; Sherman and Chole, 2001). This was assumed to result from the blocking of catecholamines but also due to inhibition of neuropeptides such as substance P and nerve growth factor (Sherman and Chole, 1995). In the present study, CALCB was significant differentially upregulated in adult IB (FC 1.8) in accordance with the suggested role of this neuropeptide as an inhibitor of osteoclast resorption (Yamamoto et al., 1986).
The bulla of the rat is a hollow bony structure. During development the growing bulla maintains a constant 60 µm wall thickness. Consequently a dynamic equilibrium exists between the resorbing osteoclasts acting on IB and the forming osteoblasts acting on OB. The signals that link and adjust resorption and formation on opposing bony surfaces are unknown. Forming and resorbing signals might be generated on OB and IB, respectively. Alternatively, all rate controlling signals might originate only on one side, either the outside or the inside to coordinate cellular activities on both sides simultaneously through short- and medium range signaling. In this study no differentially expressed single genes were identified on the OB. This finding favors a location of the basic rate controlling growth signal at the IB. Theoretically, a short range resorptive signal might control bone resorption on the inner bullar surface, while a coupled medium range formative signal might reach the outer surface through the bullar wall via the lacuno-canalicular porosity or the cellular osteocytic network at a 60 µm distance (Drossopoulou et al., 2000). This may explain how a constant wall thickness is maintained even when the rate of resorption varies.
The present study demonstrated differential expression of osteoblast related proteins like S100A8, S100A9, and FBN. It also showed osteoclast related proteins like; MMP10, TNN, S100A8, S100A9, CAPN6, and CALCB on the IB surface. Some of the factors have previously been reported in the middle ear literature (Amar et al., 1996; Lang et al., 1997; Kwon et al., 2006; Juhasz et al., 2009) while others are new candidate genes in MEACS modeling. Furthermore, the novel gene sets “osteoclast related genes”, “osteoblast related genes” and the formative genes comprising the public gene set “BONE_REMODELING” were also significantly enriched on the IB. This indicates that the controlling signals originate on the inside and that the genes work in functional groups.
Differentially expressed signals may be overlooked for a number of reasons. Individual osteoclasts are very effective and the process of resorption may proceed at a high rate with only a few active osteoclasts involved. Besides, important bone-related genes may be lost through attenuation by dilution as they may be found only in the smaller fraction of the lining tissue, which represents the osteoclast domain in close contact with the bone surface. Future isolation of such microscopic subpopulations of lining cells may be possible with the laser microdissection technique (Markaryan et al., 2011).
Furthermore, signaling proteins may be secreted in a cyclical pattern rather than continually and some signaling proteins may reach the lining cells via an opposite route from within the lacuna–canalicular system.
Finally, in this study the levels of mRNA expression determined were assumed to correspond to the levels of protein abundance (transcriptional control). In the pathway from DNA to RNA to protein the gene expression can be regulated at many steps. Some studies show results with limited correlations between mRNA and protein expression levels (Greenbaum et al., 2003). This may result from another level of regulation of gene expression or because of stochastic noise in mRNA and protein expression experiments. However, for most genes transcriptional controls are thought to be paramount as only this kind of control ensures that no superfluous intermediates are synthesized.
The qPCR validation confirmed all the microarray results except for RANKL. The qPCR result of RANKL was contrary to the array data significant. Thus, independent validation of microarray data continues to be an important consideration.
The present study of lining tissue in the normal developing bulla identified several differentially expressed genes and gene sets of potential significance. These factors and gene sets may also play important roles during pathologic pneumatization of the middle ear air cell system in common middle ear diseases. Furthermore, resorptive as well as formative signals may all originate from IB lining cells. Future array studies on young animals with experimental otitis compared to the present results may offer important clinical information by demonstrating which of the differentially expressed gene products are responsible for the decreased pneumatization caused by otitis media. Furthermore, future studies should include other methodologies, for example, miRNA and proteomics analyses. This may reveal more information about the bone metabolism in the MEACS and verify the observed changes of gene expression in this study.