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Correspondence to: Stephen P. Evanko, U. of Washington School of Medicine, Dept. of Pathology, Box 357470, Seattle, WA 98195.
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Summary |
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Hyaluronan is a high molecular weight glycosaminoglycan found in the extracellular matrix of many tissues, where it is believed to promote cell migration and proliferation. It was recently shown that hyaluronan-dependent pericellular matrix formation is a rapid process that occurs as cells detach during mitosis. Growing evidence for intracellular hyaluronan in tissues in vivo, together with evidence of intracellular hyaluronan binding molecules, prompted us to examine hyaluronan distribution and uptake as well as hyaluronan binding sites in cells and their relationship to cell proliferation in vitro, using a biotinylated hyaluronan binding protein and fluorescein-labeled hyaluronan. In permeabilized smooth muscle cells and fibroblasts, hyaluronan staining was seen in the cytoplasm in a diffuse, network-like pattern and in vesicles. Nuclear hyaluronan staining was observed and confirmed by confocal microscopy and was often associated with nucleoli and nuclear clefts. After serum stimulation of 3T3 cells, there was a dramatic increase in cytoplasmic hyaluronan staining, especially during late prophase/early prometaphase of mitosis. In contrast, unstimulated cells were negative. There was a pronounced alteration in the amount and distribution of hyaluronan binding sites, from a mostly nucleolar distribution in unstimulated cells to one throughout the cytoplasm and nucleus after stimulation. Exogenous fluorescein-labeled hyaluronan was taken up avidly into vesicles in growing cells but was localized distinctly compared to endogenous hyaluronan, suggesting that hyaluronan in cells may be derived from an intracellular source. These data indicate that intracellular hyaluronan may be involved in nucleolar function, chromosomal rearrangement, or other events in proliferating cells. (J Histochem Cytochem 47:13311341, 1999)
Key Words: hyaluronan, glycosaminoglycans, mitosis, cell proliferation, nuclear matrix, nucleoli
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Introduction |
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Hyaluronan is a high molecular weight glycosaminoglycan generally regarded as an extracellular matrix component that facilitates cell locomotion and proliferation, and plays a role in wound healing and in developmental processes such as cell condensation and migration. The role of hyaluronan in cell proliferation is well documented (
In addition to its role in the extracellular matrix, evidence is growing that hyaluronan is also present in the cytoplasm and nuclei of cells in a number of tissues in vivo (
This study examined the intracellular localization of hyaluronan and hyaluronan binding sites, as well as hyaluronan uptake, and their relationship to cell proliferation in vitro. The results show that there is an increase in the amount of intracellular hyaluronan and a redistribution of hyaluronan binding sites that are associated specifically with mitosis, and provide evidence that cytoplasmic and nuclear hyaluronan may be derived from intracellular sources.
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Materials and Methods |
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Cell Culture
Human arterial smooth muscle cells (SMCs) derived from explants of human aortic tissue were kindly provided by Dr. Russell Ross (Department of Pathology, University of Washington). Human skin fibroblasts were provided by Dr. Peter Byers (Department of Pathology, University of Washington). Swiss 3T3 D1 cells were provided by Dr. Daniel BowenPope (Department of Pathology, University of Washington). All cells were maintained in DMEM containing 10% fetal bovine serum. For cytochemical localization, cells were seeded on coverslips in 35-mm tissue culture dishes at 2 x 105 cells per dish. In certain experiments, 3T3 cells or SMCs were made quiescent by switching the medium to DMEM containing 0.5% fetal bovine serum for 48 hr and were then stimulated by readdition of serum to 10% or addition of 10 ng/ml PDGF AB. Cells were prepared for hyaluronan localization as described below at 12 hr, 24 hr, and 27 hr after serum addition to correlate with the times that cells are expected to be in G1, S, and G2/M of the cell cycle (not shown). To examine hyaluronan internalization, fluorescein-labeled hyaluronan (1.65 µg/ml) was added to the medium of stimulated and unstimulated cells after serum addition and was incubated for 24 hr. Cells were then stained for endogenous hyaluronan as described below.
Localization of Hyaluronan and Hyaluronan Binding Sites
Cells on coverslips were digested with 2 U/ml Streptomyces hyaluronidase (Sigma; St Louis, MO) in DMEM at 37C for 1 hr before fixation to remove pericellular hyaluronan, which interfered with the visualization of intracellular hyaluronan. After rinsing with PBS, the cultures were fixed by addition of 10% neutral buffered formalin directly to the medium to give a final concentration of 2.5% formalin, for 10 min at 22C. Cells were rinsed three times with PBS and permeabilized with 0.5% Triton X-100 in PBS for 10 min. To localize hyaluronan, cells were stained with a biotinylated probe consisting of a mixture of cartilage proteoglycan core protein and link protein (kindly provided by Charles Underhill; Department of Anatomy and Cell Biology, Georgetown University) at a concentration of 2 µg/ml. This is a commonly used probe that specifically recognizes hyaluronan in tissues and cultured cells (
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To compare the distribution of endogenous hyaluronan with exogenous hyaluronan taken up by cells by endocytosis, 3T3 cells on coverslips were made quiescent by incubation in medium containing 0.2% fetal bovine serum for 48 hr, and then some cultures were stimulated to proliferate by addition of serum to 10%. Fluorescein-labeled hyaluronan was added (5 µg/ml) and cells were allowed to take up the labeled hyaluronan for 24 hr. Cells were then fixed and stained for endogenous intracellular hyaluronan using the biotinylated probe and streptavidinTexas Red.
To localize intracellular hyaluronan binding sites, fixed and permeabilized cells were incubated with fluorescein-labeled hyaluronan (5 µg/ml) for 10 min. after having stained the endogenous intracellular hyaluronan with Texas Red. As a control, binding of the labeled hyaluronan was blocked by preincubation of the cells with excess unlabeled hyaluronan (3 mg/ml). Additional controls included digestion of the fluoresceinhyaluronan with Streptomyces hyaluronidase (20 U/ml in PBS, 37C, 18 hr), chondroitinase ABC (10 U/ml, 37C, 18 hr), or pronase (200 µg/ml, 37C, 18 hr), preincubation and staining of the cells in the presence of chondroitin sulfate (10 mg/ml), and staining of the cells with free fluoresceinamine (see Figure 7). Cells were examined with a Zeiss Axioskop photomicroscope equipped for epifluorescence or with an ACAS Ultima confocal laser scanning microscope with UV and 488-nm excitation to view DAPI and FITC, respectively.
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Preparation of Fluorescein-labeled Hyaluronan
Hyaluronan was labeled with fluorescein according to published methods (
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Results |
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Intracellular Localization of Hyaluronan
A biotinylated hyaluronan binding probe was used for localization of hyaluronan in human aortic smooth muscle cells and skin fibroblasts in vitro. In nonpermeabilized cells, hyaluronan staining was prominent on the cell surface and in the pericellular matrix as a tangled flocculent network (Figure 1A). Digestion of the cells with Streptomyces hyaluronidase before fixation abolished the pericellular matrix and cell surface staining (Figure 1B). Occasionally, a small amount of residual punctate staining of the cell surface was visible. Cells that had been pretreated with Streptomyces hyaluronidase to remove extracellular hyaluronan were permeabilized by treatment with 0.5% Triton X-100 and then stained with the hyaluronan probe. In the permeabilized cells, there was prominent cytoplasmic staining, which appeared both in round vesicular structures, probably representing endocytotic vesicles (see below), and also in a diffuse pattern throughout the cytoplasm (Figure 1C). The cytoplasmic staining was often particularly prominent in the perinuclear region. In SMC cultures, approximately 75% of the cells exhibited positive cytoplasmic staining. However, there were marked variations in the intensity of staining among hyaluronan-positive cells. Interestingly, cells having similar staining intensities were often seen in small groupings (not shown). Digestion of the cells with hyaluronidase a second time, after permeabilization, entirely abolished the intracellular staining, confirming the specificity of the hyaluronan probe (Figure 1D). Preincubation of the hyaluronan binding probe with excess hyaluronan also abolished staining (not shown).
In addition to cytoplasmic staining, apparent nuclear localization was also frequently seen. In SMCs, the nuclear staining often appeared to be associated with nucleoli (Figure 1C; see also below). Hyaluronan staining occasionally could also be seen as fine filamentous material throughout the nuclei, but this was difficult to resolve on photomicrographs.
The hyaluronan localization described above employed a diffusable chromogenic substrate. One concern was that the diffuse staining in the cytoplasm, as well as the perinuclear and nucleolar staining, resulted from diffusion of the chromogen away from the cytoplasmic vesicles. Therefore, we used streptavidin conjugated to Texas Red or FITC as a nondiffusable marker to localize the biotinylated hyaluronan binding probe. An identical hyaluronidase-sensitive pattern was observed with the fluorescent markers (Figure 2), indicating that the fine network-like cytoplasmic staining observed with the peroxidase substrate did not result from diffusion.
In permeabilized human skin fibroblasts, hyaluronan staining in the cytoplasm was seen in a similar pattern as in the SMCs, i.e., both vesicular and diffuse cytoplasmic staining (Figure 3). However, there was a noticeably different pattern of nuclear staining in the fibroblasts. In addition to occasional nucleolar staining, there was a clear association of hyaluronan staining with distinct clefts and furrows in the fibroblast nuclei.
Although many examples of distinct nucleolar staining were observed, it was possible that this was actually hyaluronan in vesicles positioned over or under the nucleus. However, confocal microscopy, which resolves the fluorescent signal in adjacent 1.0-µm-thick optical sections, demonstrated that the hyaluronan staining (green label) was in the same focal plane as the nuclear DAPI staining, confirming the nuclear localization (Figure 4).
Possible Intracellular Source of Hyaluronan
To determine whether the cytoplasmic and/or nuclear hyaluronan resulted from translocation of extracellular hyaluronan or if it might be derived from intracellular sources, either quiescent, serum-starved 3T3 cells or cells stimulated with 10% fetal bovine serum were allowed to take up fluorescein-labeled hyaluronan for 24 hr and then were fixed, permeabilized, and stained for intracellular hyaluronan with Texas Redstreptavidin. In the quiescent control cells, uptake of fluorescein-labeled hyaluronan was minimally detectable, was in small vesicles, and was seen only in a few scattered cells (Figure 5A). Interestingly, hyaluronan staining with the hyaluronan binding probe in the same control cells was only occasionally detectable and was confined to the same vesicles that contained the fluorescent hyaluronan, but was negative in the cytoplasm and nuclei. This indicates that fluorescein remains associated with hyaluronan after internalization and further confirms the specificity of the hyaluronan binding probe. This also supports the idea that the positively stained vesicular structures shown in Figure 1 contain internalized endogenous hyaluronan. By contrast, in the serum-stimulated cells there were both prominent cytoplasmic staining for hyaluronan and increased uptake of fluorescein-labeled hyaluronan into endosomal vesicles (Figure 5B). However, the exogenous fluoresceinated hyaluronan was confined to the endosomes and did not co-localize with the endogenous hyaluronan network in the cytoplasm, suggesting that some of the cytoplasmic hyaluronan may be derived from intracellular sources and not from translocation of extracellular hyaluronan. Interestingly, uptake of the fluoresceinated hyaluronan occurred to the greatest extent in cells with a stellate or motile fusiform morphology, consistent with a recent study showing rapid uptake of hyaluronan in motile cells (
Distribution of Hyaluronan Binding Sites
In a related experiment, the fluorescein-labeled hyaluronan was used as a probe to localize intracellular hyaluronan binding sites in permeabilized cells. In this case, fixed and permeabilized cells were first stained for endogenous intracellular hyaluronan using the biotinylated hyaluronan binding probe and Texas Redstreptavidin, and then incubated with fluorescein-labeled hyaluronan. In the quiescent control cells, fluorescein-labeled hyaluronan bound to a fine, lace-like network in the cytoplasm and, most prominently, to nucleoli in the nucleus (Figure 5C and Figure 5G). As above, there was little or no staining for endogenous hyaluronan. Incubation of the cells with excess unlabeled hyaluronan (3 mg/ml) dramatically diminished binding of the labeled hyaluronan (Figure 5D and Figure 6C). Additional controls confirmed the specificity of this binding (Figure 6). For example, chondroitin sulfate had negligible effect on the binding, even at a threefold higher concentration (10 mg/ml). In addition, exhaustive digestion of the labeled hyaluronan with Streptomyces hyaluronidase (Figure 6) or chondroitinase ABC (not shown) dramatically diminished the binding to permeabilized cells. Pronase digestion of the fluoresceinhyaluronan preparation had no effect, indicating that the binding was not due to any protein contaminants in the preparation. In addition, no binding was seen with free fluorescein.
In contrast to quiescent cells, there was a pronounced increase in the binding of fluorescein-labeled hyaluronan to the reticular network in the cytoplasm in serum-stimulated cells, indicating an increase in the amount of available binding sites (Figure 5E, Figure 5F, and Figure 5H). In stimulated cells, the bound fluoresceinhyaluronan (green) co-localized to a large extent with the staining of the endogenous hyaluronan network (red label) in the cytoplasm of both premitotic (Figure 5E) and mitotic cells (Figure 5F), but not in the endosomal vesicles. Similar results were seen in fibroblasts and SMCs (not shown). Interestingly, in mitotic cells the fluorescent hyaluronan bound to sites between the chromosomes and was similar to the distribution of endogenous hyaluronan (also see below; compare cells in prometaphase in Figure 5F and Figure 7C). Futhermore, in marked contrast to the control cells, in which the the fluorescein-labeled hyaluronan bound primarily to nucleoli, the fluorescent hyaluronan bound extensively throughout the nucleus and/or to the nuclear periphery in the stimulated cells. A higher-magnification view, shown in Figure 5G and Figure 5H, compares the extent to which fluorescein-labeled hyaluronan bound to the reticular network in the cytoplasm and to the nucleus in control vs stimulated cells. Interestingly, in many of the nuclei in the stimulated cells there were many round, uniformly sized dark spots, perhaps representing nuclear pores, that presumably were made apparent by negative staining with the fluoresceinated hyaluronan which bound to the nuclear periphery. This may represent binding to areas of condensed chromatin (
Association of Intracellular Hyaluronan with Mitosis
As described above, there appeared to be a striking increase in the amount of intracellular hyaluronan and hyaluronan binding sites in serum-stimulated cells. It was previously demonstrated that formation of hyaluronan-dependent extracellular matrix occurs in the pericellular space just before the detachment phase of mitotic cell rounding in vitro (
Virtually no intracellular hyaluronan staining was observed in serum-starved quiescent cells, i.e., G0/interphase (Figure 7A). After serum stimulation, hyaluronan staining was detectable by 12 hr, when the cells are expected to be in G1 of the cell cycle, and was seen as a diffuse network throughout the cytoplasm and concentrated around the nucleus (not shown). Staining increased gradually in almost all cells and, compared to 12 hr, was clearly stronger at 24 hr (Figure 7B), which is just after the peak of DNA synthesis in these cells. Most notably, at 27 hr there was a striking increase in the intensity of cytoplasmic hyaluronan staining specifically in the mitotic cells. In late prophase/early prometaphase, when individual chomosomes become visible and the nuclear membrane breaks down, hyaluronan staining was strong and, in several cases, appeared to extend from the periphery of the nucleus into the space between chromosomes (Figure 7C). Hyaluronan filled the cell and completely surrounded the chromosomes at the metaphase plate (Figure 7D) and then filled the space between the separating chromosomes during anaphase (Figure 7E). Strong hyaluronan staining persisted through telophase (Figure 7F). Intense staining of mitotic SMCs after stimulation by PDGF was also seen (not shown), indicating that this is a general effect of mitogenic stimulation and not an undefined serum effect. These observations indicate that hyaluronan may have a functional role on the inside of the cells as well as in the pericellular matrix, specifically during mitosis.
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Discussion |
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Hyaluronan is a structurally simple yet evolutionarily ancient molecule, which has a number of diverse functions (
The presence of intracellular hyaluronan may be a general phenomenon because we have also found it in bovine endothelial cells, human mammary epithelial cells, and mammary tumor cells (unpublished observations.) However, the source of cytoplasmic and nuclear hyaluronan is not clear. The present results suggest that in 3T3 cells, fibroblasts, and SMCs, some of the cytoplasmic and nuclear hyaluronan may be derived from an intracellular source. However, this is not yet fully resolved. One recent study reported rapid uptake and translocation of labeled hyaluronan to the nucleus and cytoplasm in association with increased cell motility (
There is increasing evidence for the presence of intracellular glycosaminoglycans, and the localization of hyaluronan in the cytoplasm, nucleus, and caveolae in tissues such as brain, liver, artery, cumulus cells, and oocytes has been reported previously (
The intracellular function(s) of hyaluronan is not yet clear, however. The observations of nucleolar hyaluronan staining and the binding of exogenous hyaluronan to nucleoli and the nuclear periphery are consistent with a study in which hyaluronan was localized to nucleoli and to areas of condensed chromatin in the nuclear periphery in oocytes and cumulus cells (
On the other hand, it is known that nucleolar components are located at the tips of certain chromosomes and that nucleolar proteins disperse to surround all of the chromosomes during mitosis, and then help reestablish the nucleoli in the daughter cells during telophase (
Precedent for regulatory roles of intracellular glycosaminoglycans comes from studies showing that nuclear heparan sulfate is involved in growth control and transcriptional regulation (
Therefore, hyaluronan now appears to be among a growing list of secreted molecules that also have an intracellular or nuclear function (
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Acknowledgments |
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We would like to thank Russell Ross, Peter Byers, and Daniel BowenPope (all of the Department of Pathology, University of Washington) for providing human smooth muscle cells, human skin fibroblasts, and 3T3 cells, respectively, and Charles Underhill (Department of Anatomy and Cell Biology, Georgetown University) for providing the hyaluronan binding probe. We also thank Terry Kavanaugh (Department of Environmental Health, University of Washington) and Stephanie Lara (Department of Pathology, University of Washington) for assistance with the confocal microscopy.
Received for publication February 5, 1999; accepted May 4, 1999.
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