E11/gp38 selective expression in osteocytes: regulation by mechanical strain and role in dendrite elongation - PubMed (original) (raw)

E11/gp38 selective expression in osteocytes: regulation by mechanical strain and role in dendrite elongation

Keqin Zhang et al. Mol Cell Biol. 2006 Jun.

Abstract

Within mineralized bone, osteocytes form dendritic processes that travel through canaliculi to make contact with other osteocytes and cells on the bone surface. This three-dimensional syncytium is thought to be necessary to maintain viability, cell-to-cell communication, and mechanosensation. E11/gp38 is the earliest osteocyte-selective protein to be expressed as the osteoblast differentiates into an osteoid cell or osteocyte, first appearing on the forming dendritic processes of these cells. Bone extracts contain large amounts of E11, but immunostaining only shows its presence in early osteocytes compared to more deeply embedded cells, suggesting epitope masking by mineral. Freshly isolated primary osteoblasts are negative for E11 expression but begin to express this protein in culture, and expression increases with time, suggesting differentiation into the osteocyte phenotype. Osteoblast-like cell lines 2T3 and Oct-1 also show increased expression of E11 with differentiation and mineralization. E11 is highly expressed in MLO-Y4 osteocyte-like cells compared to osteoblast cell lines and primary osteoblasts. Differentiated, mineralized 2T3 cells and MLO-Y4 cells subjected to fluid flow shear stress show an increase in mRNA for E11. MLO-Y4 cells show an increase in dendricity and elongation of dendrites in response to shear stress that is blocked by small interfering RNA specific to E11. In vivo, E11 expression is also increased by a mechanical load, not only in osteocytes near the bone surface but also in osteocytes more deeply embedded in bone. Maximal expression is observed not in regions of maximal strain but in a region of potential bone remodeling, suggesting that dendrite elongation may be occurring during this process. These data suggest that osteocytes may be able to extend their cellular processes after embedment in mineralized matrix and have implications for osteocytic modification of their microenvironment.

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Figures

FIG. 1.

FIG. 1.

Identification of a 40-kDa protein highly expressed in osteocytes. With monoclonal antibody 9C11, made against MLO-Y4 cells, a 40-kDa band was identified by Western blotting and shown to be highly expressed in MLO-Y4 osteocyte-like cells. The top part of each panel is a Western blot assay, and the bottom is a Ponceau S-stained gel to show relative amounts of loaded protein. Theoretically, as osteoblasts form a mineralized matrix, any cells trapped in that matrix would have the characteristics of osteocytes. Under mineralizing culture conditions, the Oct-1 cells began to express this antigen at 4 weeks (A) and the 2T3 cells began to express this antigen earlier and in large amounts, at 3 and 4 weeks of culture (B). The circles are von Kossa-stained 2T3 cultures showing increased mineralization with time. This suggests that these cells are differentiating into osteocytes in culture. The 9C11 antibody also recognized a band in bone extracts (C). The 40-kDa band is present in MLO-Y4 cells but was not present in osteoblast-like cells such as MC3T3 (MC) and Oct-1 cells (Oct), nor was it visible in extracted cells. These cells were isolated from 6-week-old mouse long bone through serial digestions of collagenase with and without EDTA (F1, F2, F3, and F4). Only the bone particles (BP) containing embedded osteocytes showed the 40-kDa band. Note the relative amounts of protein in the particle fraction compared to protein in the cell fractions. Similar experiments were repeated with the 8.1.1 antibody (D). The Western blot assay is on the left, and the Ponceau stain is on the right. F1 to F6 represent cells on the bone surface that are removed by serial digestions with collagenase. F7 and F8 represent cells on the bone surface that could be removed by EDTA, followed by collagenase. Bone particles (BP) represent the remaining bone that was subjected to boiling in SDS sample buffer. Note that considerably less protein was loaded in the well containing the bone particle extracted protein.

FIG. 2.

FIG. 2.

Primary osteoblasts are negative for E11 expression but begin to express this antigen with time in culture. MLO-Y4 osteocyte-like cells express the highest levels of E11. E11 in primary periosteal osteoblasts is not detectable upon isolation but increases with time in culture (A). Cells were isolated from long bones of 1-week-old mice and either lysed after isolation or cultured for 3 to 7 days before processing for Western blot analysis. The left part shows the reaction with the 8.1.1 antibody, and the right part shows Ponceau S staining. Note the lack of expression in the freshly isolated cells but the increased expression with time in culture. Immunocytochemical staining for E11 in freshly isolated cytocentrifuged mouse osteoblasts (B) and osteoblasts cultured for 3 days and then subjected to collagenase treatment and cytocentrifuged (C) was performed. This experiment was performed to show that collagenase treatment of the freshly isolated cells or cultured cells was not removing or having an effect on E11 expression on the cell surface. The insert in the upper right part shows the negative control. Scale bar = 200 μm. E11 expression is much higher in the MLO-Y4 osteocyte-like cell line than in other cell lines, as determined by Western blotting (D). The osteocyte-like cell line MLO-Y4 (Y4), osteoblast-like cell lines MC3T3 (MC) and 2T3, late osteoblast-early osteocyte MLO-A5 (A5) cells, primary osteoblasts (OB), primary fibroblasts (FB), and the fibroblast-like cell line NIH 3T3 (NIH) were cultured for 3 to 4 days before lysis. The upper and middle parts show reaction with the 8.1.1 antibody. The film was exposed for a short period of time to visualize relative expression (top) and for a longer period of time to visualize any additional bands (middle). The lower part shows Ponceau S staining. Note that the highest expression was in the MLO-Y4 osteocyte-like cells compared to less or no expression in the other cell lines. Bands of various sizes were observed, suggesting different extents or forms of posttranslational modification. A 100-kDa band can be seen with the MLO-Y4 cell, MLO-A5 cell, and primary osteoblast lysates after longer exposure.

FIG. 3.

FIG. 3.

Immunostaining for E11 is only observed in osteocytes, not osteoblasts, cartilage, growth plate, or marrow, in vivo. Localization of E11 in kidney, lung, and brain tissues is shown. Immunohistochemical staining for E11 with the 8.1.1 antibody in a 19-day-old C57BL/6 mouse tibia. Positive staining is brown. The positive osteocytes (arrows) can be seen within the trabecular bone but not in the growth plate (A) and within the cortical bone but not in the marrow (B). Higher magnification of the cortical bone shows that the osteocytic cell body and dendrites are positive, while the cells on the surface are negative (C). Immunohistochemical staining of E11 can be observed in kidney glomeruli because of podocytes (D), in the lung because of type 1 alveolar cells (E), and in the choroid plexus of the brain (F). It is known that E11 is expressed in the podocyte in the kidney, where it is known as podoplanin (3), and in type 1 alveolar lung cells, where it is referred to as T1alpha/RT140 (15, 39). No staining was observed in liver or muscle tissue (data not shown). The upper left insert in panels A, B, D, E, and F shows the negative control with normal hamster IgG in place of the primary antibody.

FIG. 4.

FIG. 4.

Fluid flow shear stress increases mRNA for E11 in MLO-Y4 cells and mineralizing 2T3 cells. Northern blotting of E11 mRNA in MLO-Y4 cells after exposure to fluid flow shear stress at 4 and 16 dynes/cm2 showed a twofold increase at 2 h after shear stress but not at 24 h, indicating that E11 is an early-response gene. The data represent an average of two experiments as shown on the graph. E11 is also an early-response gene in mineralizing 2T3 cells, as shown by an increase at 2 h after exposure to fluid flow shear stress at 16 dynes/cm2, compared to OPN (osteopontin), which is a late-responding gene increased at 24 h, not at 2 h, after shear stress. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIG. 5.

FIG. 5.

Length of dendritic processes of MLO-Y4 cells is increased in response to mechanical strain in vitro. This elongation is blocked by siRNA specific for E11. MLO-Y4 cells stained with crystal violet without fluid flow (part C) and with exposure to 16 dynes/cm2 for 2 h, followed by 24 h of incubation (part FF), show increased length of dendrites (A). The length of the dendrites is significantly increased. The formation of dendritic processes in response to shear stress is blocked by siRNA specific for E11 (B). A combination of three siRNAs specific to E11 when added for 24 h of incubation before 2 h of fluid flow shear stress inhibited the elongation of dendritic processes. No effect was observed with the RISC-free siRNA or the vehicle (Veh). The experiments were repeated three to six times and combined for statistical evaluation. Western blot analysis (C) showed that E11 protein expression was reduced approximately 50 to 70% in these cells. **, significantly different from control (P < 0.005).

FIG. 6.

FIG. 6.

Generation and characterization of _E11_-null mice. A schematic of the mouse E11 wild-type (WT) locus with locations of key restriction enzyme sites and the lacZpA vector with the neo cassette inserted into exon 1 and intron 1 at the SalI and SmaI sites is shown. In the heterozygotes, one of the E11 alleles is replaced with the inserted lacZ cDNA, used to reflect the endogenous E11 expression pattern. Immunohistochemical staining for E11 protein expression was present in the osteocytes in the long bones of wild-type (+/+) embryos (B) but not _E11_-null (−/−) embryos (A), as shown by the brown staining (arrow). Scale bar = 100 μm. Hematoxylin-and-eosin (H&E)-stained sections of femurs isolated from _E11_-null and wild-type mouse embryos are compared in panels C, D, E, and F. Histological measurements are shown in Table 1. No significant differences were observed between long bones from _E11_-null and wild-type embryos. LacZ staining in the E11 heterozygote (+/−) localizes with protein staining in the osteocyte but does not correspond to E11 protein expression in other tissues, such as lung tissue. (G) Background LacZ staining in the 12.5-dpc wild-type embryo. (H) LacZ staining in the 12.5-dpc embryo in the dorsal spinal chord, in the lung bud, and in an area of the ventral spinal chord. Osteocytes in the rib are positive for LacZ (I) (scale bar = 0.2 μm), whereas there is a lack of LacZ staining in the lung (J) from the same newborn (scale bar = 0.5 μm). Lack of LacZ staining in the lung persisted as shown at 3 weeks of age (L) (scale bar = 0.5 μm) but continued in osteocytes as shown in the tails of heterozygote mice (K) (scale bar = 20 μm). KO, knockout; β-gal, β-galactosidase.

FIG. 7.

FIG. 7.

Analysis of the E11 gene sequence (accession no. AY115493) used for these studies. AY115493 is the sequence we submitted and published in GenBank from the 129 mouse strain. NT_094216 (MM4_93853_34, genome) is a Mus musculus strain 129 chromosome 4 genomic contig. NT_039267 (MM4_39307_34) is a M. musculus strain C57BL/6J chromosome 4 genomic contig. AC098724 is M. musculus strain C57BL/6J bacterial artificial chromosome clone RP23-3D14 from chromosome 4. AL611982 is a mouse DNA sequence from clone RP23-348F1 on chromosome 4, also from strain C57BL/6J. Therefore, the C57BL/6J strain contains an SmaI cleavage site at this position in the promoter whereas the 129 mouse strain does not. Sequence analysis with the Transcription Element Search System revealed Ets-2 and Pit-1a binding sites in part of the deleted sequence. Several Sp1 sites and an Ap-1 site are also present in the deleted sequence, nucleotides 9680 to 10137. The nucleotide position numbering is that in the original GenBank sequence file. (R) indicates that the transcription factor recognizes the reverse complement sequence.

FIG. 8.

FIG. 8.

Mechanical loading of mouse ulnae shows an increase in E11 expression as determined by both lacZ expression and immunostaining for E11 protein. Maximal expression is not observed in areas of maximal strain. E11-lacZ heterozygote mice and wild-type mice at 3 months of age were loaded at 3.5 N for 60 cycles at 2 Hz. The values shown were obtained 4 h after loading for lacZ expression and 24 h after loading for E11 expression. Cross sections of ulnae taken at 6.5 mm from the olecranon show X-Gal staining of osteocytes in a loaded ulna (A) and less staining in a nonloaded control ulna (B). Higher magnifications (C and D) are from the boxed areas of panels A and B, respectively, showing blue staining within osteocytes. The loaded and unloaded sections in panels E and F, respectively, are representative of immunohistochemical staining for E11 protein (brown). The graphs show the percentage of positive osteocytes (y axis) in each section, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, and 10.5 mm from the olecranon (x axis). A significant increase in E11 expression was observed in the sections 4.5, 5.5, and 6.5 mm from the olecranon. Note that osteocytes within the bone matrix also showed increased expression in response to loading (n = 4 or 5 for lacZ, n = 3 for immunostaining). Data are the mean ± the standard error of the mean. *, P < 0.05; **, P < 0.01. Previously we have noted elevated expression of genes such as those for Dmp1 and MEPE in areas of maximal strain, 8.5 to 10.5 mm from the olecranon (20, 26) (red). However, elevated E11 expression was observed in a region where increased resorption occurs in response to mechanical loading, 4.5 to 6.5 mm from the olecranon (blue). This suggests that increased E11 expression in osteocytes in this area may be associated with remodeling.

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