Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs

Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry-tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)-procollagen N-terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO-A5 osteoblast-like cells and fibronectin-null mouse embryonic fibroblasts (FN-null-MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co-precipitated with α1(I)-collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen-containing extracellular fibrils in the presence of ascorbate. Immunogold-EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO-A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell-generated forces can physically reshape the collagen fibrils. Using co-cultures of mCherry- and GFPtpz-collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno-EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN-null-MEFs expressing GFPtpz-collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP-collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen

Hemorrhage activates proopiomelanocortin neurons in the rat hypothalamus

Severe blood loss lowers arterial pressure through a central mechanism that is thought to include opioid neurons. In this study, we investigated whether hemorrhage activates proopiomelanocortin (POMC) neurons by measuring Fos immunoreactivity and POMC mRNA levels in the medial basal hypothalamus. Hemorrhage (2.2 ml/100 g body weight over 20 min) increased the number of Fos immunoreactive neurons throughout the rostral-caudal extent of the arcuate nucleus, the retrochiasmatic area and the peri-arcuate region lateral to the arcuate nucleus where POMC neurons are located. Double label immunohistochemistry revealed that hemorrhage increased Fos expression by beta-endorphin immunoreactive neurons significantly. The proportion of beta-endorphin immunoreactive neurons that expressed Fos immunoreactivity increased approximately four-fold, from 11.7 +/- 1.4% in sham-operated control animals to 42.0 +/- 5.2% in hemorrhaged animals. Hemorrhage also increased POMC mRNA levels in the medial basal hypothalamus significantly, consistent with the hypothesis that blood loss activates POMC neurons. To test whether activation of arcuate neurons contributes to the fall in arterial pressure evoked by hemorrhage, we inhibited neuronal activity in the caudal arcuate nucleus by microinjecting the local anesthetic lidocaine (2%; 0.1 or 0.3 mu l) bilaterally 2 min before hemorrhage was initiated. Lidocaine injection inhibited hemorrhagic hypotension and bradycardia significantly although it did not influence arterial pressure or heart rate in non-hemorrhaged rats. These results demonstrate that hemorrhage activates POMC neurons and provide evidence that activation of neurons in the arcuate nucleus plays an important role in the hemodynamic response to hemorrhage

Time Lapse Imaging Techniques for Comparison of Mineralization Dynamics in Primary Murine Osteoblasts and the Late Osteoblast/Early Osteocyte-Like Cell Line MLO-A5

Mineralization of bone matrix and osteocyte differentiation occur simultaneously and appear interrelated both spatially and temporally. Although these are dynamic events, their study has been limited to using static imaging approaches, either alone or in combination with chemical and biochemical analysis and/or genetic manipulation. Here we describe the application of live cell imaging techniques to study mineralization dynamics in primary osteoblast cultures compared to a late osteoblast/early osteocyte-like cell line, MLO-A5. Mineral deposition was monitored using alizarin red as a vital stain for calcium. To monitor differentiation into an osteocyte-like phenotype, the calvarial cells were isolated from transgenic mice expressing green fluorescent protein (GFP) driven by an 8-kb dentin matrix protein-1 (Dmp1) promoter that gives osteocyte-selective expression. Time lapse imaging showed that there was a lag phase of 15–20 h after β-glycerophosphate addition, followed by mineral deposition that was rapid in primary osteoblast cultures but more gradual in MLO-A5 cultures. In primary osteoblast cultures, mineral was deposited exclusively in association with clusters of cells expressing Dmp1-GFP, suggesting that they were already differentiating into osteocyte-like cells. In MLO-A5 cells, the first indication of mineralization was the appearance of punctate areas of alizarin red fluorescence of 4–7 μm in diameter, followed by mineral deposition throughout the culture in association with collagen fibrils. A high amount of cell motility was observed within mineralizing nodules and in mineralizing MLO-A5 cultures. These studies provide a novel approach for analyzing mineralization kinetics that will enable us to dissect in a time-specific manner the essential players in the mineralization process

A Novel Osteogenic Cell Line that Differentiates into GFP‐Tagged Osteocytes and forms Mineral with a Bone‐like Lacunocanalicular Structure

Osteocytes, the most abundant cells in bone, were once thought to be inactive but are now known to have multifunctional roles in bone, including in mechanotransduction, regulation of osteoblast and osteoclast function and phosphate homeostasis. Because osteocytes are embedded in a mineralized matrix and are challenging to study, there is a need for new tools and cell models to understand their biology. We have generated two clonal osteogenic cell lines, OmGFP66 and OmGFP10, by immortalization of primary bone cells from mice expressing a membrane‐targeted GFP driven by the Dmp1‐promoter. One of these clones, OmGFP66, has unique properties compared to previous osteogenic and osteocyte cell models and forms 3‐dimensional mineralized bone‐like structures, containing highly dendritic GFP‐positive osteocytes, embedded in clearly defined lacunae. Confocal and electron microscopy showed that structurally and morphologically, these bone‐like structures resemble bone in vivo, even mimicking the lacunocanalicular ultrastructure and 3D spacing of in vivo osteocytes. In osteogenic conditions, OmGFP66 cells express alkaline phosphatase, produce a mineralized type‐I‐collagen matrix and constitutively express the early osteocyte marker, E11/gp38. With differentiation they express osteocyte markers, Dmp1, Phex, Mepe, Fgf23 and the mature osteocyte marker, Sost. They also express RankL, Opg and Hif1α, and show expected osteocyte responses to PTH, including downregulation of Sost, Dmp1 and Opg and upregulation of RankL and E11/gp38. Live‐cell imaging revealed the dynamic process by which OmGFP66 bone‐like structures form, the motile properties of embedding osteocytes and the integration of osteocyte differentiation with mineralization. The OmGFP10 clone showed an osteocyte gene expression profile similar to OmGFP66, but formed less organized bone nodule‐like mineral, similar to other osteogenic cell models. Not only do these cell lines provide useful new tools for mechanistic and dynamic studies of osteocyte differentiation, function and biomineralization, but OmGFP66 cells have the unique property of modeling osteocytes in their natural bone microenvironment

Gene set enrichment analysis of PTH Responses in the IDG-SW3 Osteocyte Enriched Cell model and <i>Ex Vivo</i> Cortical Bone Osteocyte Model.

<p><b>(A)</b> 33,277 gene expression values were analyzed against the entire 1794 geneset of PTH responsive genes in the IDG-SW3 cell model. Highly significant enrichment (NES -1.44) was found in the genes in both models that were negatively responsive to PTH (365, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125731#pone.0125731.s003" target="_blank">S1 Table</a>). <b>(B)</b> The 574 genes that responded positively to PTH in the IDG-SW3 cell model, was analyzed against the 33,277 gene expression values in the <i>ex vivo</i> bone cortical osteocyte model, and a highly significant enrichment (NES = +1.98) was found in a set of genes that positively responded to PTH (131 geneset, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125731#pone.0125731.s004" target="_blank">S2 Table</a>).</p

The effect of PTH on osteoblast and osteocyte marker gene and protein expression in IDG-SW3 cells and primary osteocytes.

<p><b>(A)</b> Real-time PCR analysis of IDG-SW3 cells cultured over a 29 day time course and treated with 50nM PTH, or PBS control, for 24 hours at days 0, 7, 14, 21 and 28. Expression was normalized to <i>Actb</i> and is relative to day 1 control samples. <i>Kera</i> and <i>E11</i> were upregulated by PTH treatment, whereas <i>Dmp1</i>, <i>Phex</i>, <i>Mepe</i> and <i>Sost</i> expression was decreased, particularly at the later time points (n = 3±SD, *p<0.05). <b>(B)</b> Real-time PCR analysis of osteocyte-enriched bone fragments cultured in the presence of 50nM PTH, or PBS, for 24 hours. Expression was normalized to <i>Actb</i> and is relative to control samples. <i>Kera</i> and <i>E11</i> were upregulated by PTH treatment. <i>Dmp1</i>, <i>Phex</i>, <i>Mepe</i> and <i>Sost</i> expression was decreased in the primary osteocytes (n = 4±SD, *p<0.05). <b>(C)</b> IDG-SW3 cells were cultured over a 30 day time course and treated with 50nM PTH, or PBS, for 48 hours at days 0, 7, 14, 21 and 28. E11 and sclerostin expression was assessed by western blotting. <b>(D)</b> Quantitation of E11 protein expression with and without PTH treatment, relative to day 2 control samples. <b>(E)</b> GFP expression in mature (day 28) IDG-SW3 cells treated with 50nM PTH for 48 hours. Scale bar = 20μm. <b>(F)</b> Quantification of GFP in IDG-SW3 cells cultured over a 30 day time course and treated with 50nM PTH for 48 hours at days 0, 7, 14, 21 and 28 (n = 4±SD, *p<0.05).</p

Fold change values of motility-associated genes upregulated by PTH treatment in IDG-SW3 cells and corresponding values in <i>ex vivo</i> cortical bone osteocytes as determined by David analysis.

<p>The full geneset can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125731#pone.0125731.s004" target="_blank">S2 Table</a>.</p><p>Fold change values of motility-associated genes upregulated by PTH treatment in IDG-SW3 cells and corresponding values in <i>ex vivo</i> cortical bone osteocytes as determined by David analysis.</p

The calcium channel blockers Diltiazem and Nifedipine have no effect on PTH changes in gene expression except for Keratocan.

<p>(A) Real-time PCR analysis showing effects of Diltiazem on gene expression in mature IDG-SW3 cells. No effects on PTH changes in gene expression were observed (n = 3±SD, p<0.05 relative to control (a), PTH (b), DH (c) and PTH & DH (d)). (B) Effects of Nifedipine on gene expression. No effects of Nifedipine were observed on PTH induced gene expression except that Nifedipine inhibited PTH induction of <i>Kera</i>. (n = 3±SD, p<0.05 relative to control (a), PTH (b), NI (c) and PTH & NI (d)) Expression was normalized to <i>Actb</i>.</p

The effect of PTH on IDG-SW3 cell morphology and motility.

<p>Images of GFP positive day 30 IDG-SW3 cells treated with PBS (<b>A</b>) or 50nM PTH (<b>B</b>) for 48 hours and captured by confocal microscopy. Scale bar = 25μm. (<b>C</b>) Mean velocity of mature IDG-SW3 cell motility in response to 50nM PTH over a 63 hour time course. Representative time lapse images can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125731#pone.0125731.s005" target="_blank">S1 Video</a> (n = 3 observation fields±SD, with each field containing 30–40 cells, *p<0.05) Changes in IDG-SW3 cell morphology induced by PTH, cAMP and forskolin. Effect on mature IDG-SW3 cell morphology induced by 48 hours treatment with dose response of (<b>D</b>) PTH, (<b>E</b>) 8-bromo-cAMP and (<b>F</b>) forskolin. Scale bar = 20μm.</p

L-type calcium channels mediate the effects of PTH on IDG-SW3 cell morphology and motility.

<p>(A, B) Real-time PCR analysis showing expression of the L and T-type channel subunits in IDG-SW3 cells cultured over a 29 day time course and treated with 50nM PTH for 24 hours at days 0, 7, 14, 21 and 28. Expression was normalized to <i>Actb</i> and is relative to day 1 control samples. (C) The L-type calcium channel blockers Nifedipine (250μM) and Diltiazem (100μM) inhibit the effect of 50nM PTH on mature IDG-SW3 cell morphology. (D) Mean velocity of mature IDG-SW3 cells treated with 50nM PTH alone or 50nM PTH with 100μM Diltiazem (DT) over a 46 hour time course (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125731#pone.0125731.s007" target="_blank">S3 Video</a>) (n = 3 observation fields±SD, with each field containing 30–40 cells, *p<0.05). Scale bar = 20μm.</p