E11/gp38 Selective Expression in Osteocytes: Regulation by Mechanical Strain and Role in Dendrite Elongation

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.

[1]  S. Ashrafi,et al.  The Canalicular Structure of Compact Bone in the Rat at Different Ages , 2002, Microscopy and Microanalysis.

[2]  A. Aruffo,et al.  Characterization and cloning of a novel glycoprotein expressed by stromal cells in T-dependent areas of peripheral lymphoid tissues , 1992, The Journal of experimental medicine.

[3]  L. Lanyon,et al.  The Effect of In Vivo Mechanical Loading on Estrogen Receptor α Expression in Rat Ulnar Osteocytes , 2002, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[4]  Y. Tsutsumi,et al.  Molecular cloning and characterization of antigens expressed on rat tumor vascular endothelial cells , 2000, International journal of cancer.

[5]  A. Verkman,et al.  Evidence against a role of mouse, rat, and two cloned human t1alpha isoforms as a water channel or a regulator of aquaporin-type water channels. , 1998, American journal of respiratory cell and molecular biology.

[6]  F. Hughes,et al.  Immunocytochemical investigation of the rat cementoblast phenotype. , 1993, Journal of periodontal research.

[7]  M. Williams,et al.  Monoclonal antibodies specific to apical surfaces of rat alveolar type I cells bind to surfaces of cultured, but not freshly isolated, type II cells. , 1988, Biochimica et biophysica acta.

[8]  L. Bonewald,et al.  Establishment of an Osteoid Preosteocyte‐like Cell MLO‐A5 That Spontaneously Mineralizes in Culture , 2001, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[9]  P. Mangeat,et al.  ERM proteins in cell adhesion and membrane dynamics. , 1999, Trends in cell biology.

[10]  Xi Jiang,et al.  Dentin matrix protein 1 expression during osteoblastic differentiation, generation of an osteocyte GFP-transgene. , 2004, Bone.

[11]  D. Burr,et al.  Effects of biomechanical stress on bones in animals. , 2002, Bone.

[12]  Lee G Luna,et al.  Manual of histologic staining methods of the Armed forces institute of pathology , 1968 .

[13]  L. Bonewald,et al.  Dmp1-deficient Mice Display Severe Defects in Cartilage Formation Responsible for a Chondrodysplasia-like Phenotype* , 2005, Journal of Biological Chemistry.

[14]  M. K. Knothe Tate,et al.  The osteocyte. , 2004, The international journal of biochemistry & cell biology.

[15]  L. Bonewald,et al.  Von Kossa Staining Alone Is Not Sufficient to Confirm that Mineralization In Vitro Represents Bone Formation , 2003, Calcified Tissue International.

[16]  S. Vilaró,et al.  Identification of PA2.26 antigen as a novel cell-surface mucin-type glycoprotein that induces plasma membrane extensions and increased motility in keratinocytes. , 1999, Journal of cell science.

[17]  H. Klenk,et al.  Cloning and characterization of gp36, a human mucin-type glycoprotein preferentially expressed in vascular endothelium. , 1999, The Biochemical journal.

[18]  L. Lanyon.  Osteocytes, strain detection, bone modeling and remodeling , 2005, Calcified Tissue International.

[19]  S. Hosier,et al.  Characterization of an antigenic determinant preferentially expressed by type I epithelial cells in the murine thymus. , 1992, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[20]  L. Bonewald,et al.  PGE2 Is Essential for Gap Junction-Mediated Intercellular Communication between Osteocyte-Like MLO-Y4 Cells in Response to Mechanical Strain. , 2001, Endocrinology.

[21]  H. Klenk,et al.  Identification of a 40-kDa Cell Surface Sialoglycoprotein with the Characteristics of a Major Influenza C Virus Receptor in a Madin-Darby Canine Kidney Cell Line (*) , 1995, The Journal of Biological Chemistry.

[22]  S. Krane,et al.  Osteocyte and osteoblast apoptosis and excessive bone deposition accompany failure of collagenase cleavage of collagen. , 2000, The Journal of clinical investigation.

[23]  T. Takano-Yamamoto,et al.  A three-dimensional distribution of osteocyte processes revealed by the combination of confocal laser scanning microscopy and differential interference contrast microscopy. , 2001, Bone.

[24]  M. I. Ramirez,et al.  1.3 kilobases of the lung type I cell T1α gene promoter mimics endogenous gene expression patterns during development but lacks sequences to enhance expression in perinatal and adult lung , 1999, Developmental dynamics : an official publication of the American Association of Anatomists.

[25]  L. Bonewald,et al.  MLO‐Y4 Osteocyte‐Like Cells Support Osteoclast Formation and Activation , 2002, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[26]  Matthew J. Silva,et al.  Experimental and finite element analysis of the rat ulnar loading model-correlations between strain and bone formation following fatigue loading. , 2004, Journal of biomechanics.

[27]  J. Stock,et al.  Targeted Disruption of the Osteoblast/Osteocyte Factor 45 Gene (OF45) Results in Increased Bone Formation and Bone Mass* , 2003, The Journal of Biological Chemistry.

[28]  B. Wold,et al.  Know Your Neighbors: Three Phenotypes in Null Mutants of the Myogenic bHLH Gene MRF4 , 1996, Cell.

[29]  M. I. Ramirez,et al.  Enhanced binding of Sp1/Sp3 transcription factors mediates the hyperoxia‐induced increased expression of the lung type I cell gene T1α , 2003, Journal of cellular biochemistry.

[30]  M. Detmar,et al.  Prox1, master regulator of the lymphatic vasculature phenotype , 2003, Cell and Tissue Research.

[31]  L. Bonewald,et al.  Expression of Functional Gap Junctions and Regulation by Fluid Flow in Osteocyte‐Like MLO‐Y4 Cells , 2001, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[32]  B. Lanske,et al.  Characterization and cloning of the E11 antigen, a marker expressed by rat osteoblasts and osteocytes. , 1996, Bone.

[33]  W. Ambrosius,et al.  Mechanical Loading of Diaphyseal Bone In Vivo: The Strain Threshold for an Osteogenic Response Varies with Location , 2001, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[34]  L. E. Lanyon,et al.  Noninvasive loading of the rat ulna in vivo induces a strain-related modeling response uncomplicated by trauma or periostal pressure , 1994, Calcified Tissue International.

[35]  Y. Tsutsumi,et al.  Association of CD44 with OTS-8 in tumor vascular endothelial cells. , 2000, Biochimica et biophysica acta.

[36]  M. Quintanilla,et al.  Ectopic Expression of PA2.26 Antigen in Epidermal Keratinocytes Leads to Destabilization of Adherens Junctions and Malignant Progression , 2000, Laboratory Investigation.

[37]  K. Nose,et al.  Isolation of a gene sequence induced later by tumor-promoting 12-O-tetradecanoylphorbol-13-acetate in mouse osteoblastic cells (MC3T3-E1) and expressed constitutively in ras-transformed cells. , 1990, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[38]  J. Brody,et al.  Cloning, characterization, and development expression of a rat lung alveolar type I cell gene in embryonic endodermal and neural derivatives. , 1995, Developmental biology.

[39]  D. Seldin,et al.  T1alpha, a lung type I cell differentiation gene, is required for normal lung cell proliferation and alveolus formation at birth. , 2003, Developmental biology.

[40]  C. Contag,et al.  Dissection of promoter control modules that direct Bmp4 expression in the epithelium-derived components of hair follicles. , 2002, Biochemical and biophysical research communications.

[41]  L. Bonewald,et al.  Establishment of an Osteocyte‐like Cell Line, MLO‐Y4 , 1997, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[42]  J. Wozney,et al.  Immortalized murine osteoblasts derived from BMP 2-T-antigen expressing transgenic mice. , 1996, Endocrinology.

[43]  D. Kerjaschki,et al.  Epitope-specific antibodies to the 43-kD glomerular membrane protein podoplanin cause proteinuria and rapid flattening of podocytes. , 1998, Journal of the American Society of Nephrology : JASN.

[44]  L. Bonewald,et al.  Effects of transforming growth factor β on bone nodule formation and expression of bone morphogenetic protein 2, osteocalcin, osteopontin, alkaline phosphatase, and type I collagen mRNA in long‐term cultures of fetal rat calvarial osteoblasts , 1994 .

[45]  H. Birkedal‐Hansen,et al.  The metalloproteinase MT1-MMP is required for normal development and maintenance of osteocyte processes in bone , 2005, Journal of Cell Science.

[46]  M. Kasper,et al.  Immunohistochemical investigations on the differentiation marker protein E11 in rat calvaria, calvaria cell culture and the osteoblastic cell line ROS 17/2.8 , 1999, Histochemistry and Cell Biology.

[47]  H. Pavenstädt,et al.  Cloning and expression of the mouse glomerular podoplanin homologue gp38P. , 2002, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[48]  H. Dvorak,et al.  T1α/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema , 2003, The EMBO journal.

[49]  Piero Carninci,et al.  High-efficiency full-length cDNA cloning. , 1999, Methods in enzymology.

[50]  C. Palumbo.  A three-dimensional ultrastructural study of osteoid-osteocytes in the tibia of chick embryos , 2004, Cell and Tissue Research.

[51]  R. Gonzalez,et al.  Purification and analysis of RTI40, a type I alveolar epithelial cell apical membrane protein. , 1998, Biochimica et biophysica acta.

[52]  G. Marotti,et al.  Osteocyte dendrogenesis in static and dynamic bone formation: an ultrastructural study. , 2004, The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology.

[53]  O. J. Whittemore,et al.  Vitamin D-enhanced osteocytic and osteoclastic bone resorption. , 1973, The American journal of physiology.