Intracellular Hyaluronan in Arterial Smooth Muscle Cells : Association with Microtubules, RHAMM, and the Mitotic Spindle
Hope Heart ProgramBenaroya Research Institute at Virginia Mason, Seattle, Washington (SPE,TNW) and Department of Pathology, The University of Washington, Seattle, Washington (WTP)
Correspondence to: Stephen Evanko, Hope Heart Program, Benaroya Research Institute at Virginia Mason, 1201 Ninth Avenue, Seattle, WA 98101-2795. E-mail: sevanko{at}hopeheart.org
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: hyaluronan microtubules RHAMM mitosis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous studies suggested that most of the hyaluronan in cells was small (<90 kD) and was found in vesicles of various sizes and shapes and/or other membranous compartments, and that smooth muscle cells prefer to take up low-molecular-weight hyaluronan (McGuire et al. 1987; Kan 1990
; Tammi et al. 2001
). The question remains, however, whether hyaluronan can serve an as-yet-undefined structural role within the cell. Recently the term "hyaluronasome" was proposed as a membrane-bound structure rich in hyaluronan in which coordinated synthetic and catabolic enzyme reactions occur in regulating the activities of the intracellular hyaladherins (Stern 2003
). We previously found altered levels of intracellular hyaluronan-binding sites and their dramatic redistribution following serum stimulation of smooth muscle cells (Evanko and Wight 1999
), but the identity of the binding sites was not clear. Various intracellular hyaladherins have been described, suggesting that intracellular hyaluronan may have important regulatory roles in such processes as cell cycle regulation, mitosis, cell motility, and RNA splicing (Hall et al. 1994
; Grammatikakis et al. 1995
; Deb and Datta 1996
; Collis et al. 1998
; Hofmann et al. 1998
; Zhang et al. 1998
; Assmann et al. 1999
; Haddad and Turley 2000
; Huang et al. 2000
; Maxwell and Pilarski 2000
; Hall et al. 2001
). Intracellular hyaluronan may function in these processes in a transient fashion, either following intracellular synthesis or after uptake.
Given the structural role of hyaluronan in the pericellular matrix, it is possible that hyaluronan also serves a dynamic structural role inside cells or is part of a mechanotransduction mechanism. However, the relationship of intracellular hyaluronan with cytoskeletal elements has not yet been described. The hyaluronan receptors RHAMMIHABP (Assmann et al. 1999; Maxwell and Pilarski 2000
) and CD44 (Underhill and Toole 1979
) are both known to associate with the cytoskeleton. RHAMM was first described as a cell surface hyaluronan receptor that mediates hyaluronan-induced motility (Turley and Torrance 1984
), In addition, RHAMM has also been described as a microtubule-associated protein that interacts with dynein and helps maintain spindle pole stability (Zhou et al. 2002
; Maxwell et al. 2003
). However, it is not clear whether hyaluronan is associated with RHAMM intracellularly. CD44, on the other hand, has been colocalized with hyaluronan within endosomes (Aguiar et al. 1999
). Here we present evidence for a close relationship between intracellular hyaluronan, microtubules, and RHAMM in human arterial smooth muscle cells. Their distribution around the mitotic spindle and nucleolar area suggests novel functions for hyaluronan within cells.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Therefore, we examined the effect of hyaluronan size on the intracellular distribution following uptake by smooth muscle cells (Figures 1A and 1B). High molecular weight fluorescein-hyaluronan mostly remained in larger vesicles with some translocation to the perinuclear area, as seen previously (Figures 1A and 1C). In contrast, when the fluorescein-hyaluronan was partially fragmented to less than 50 kD by digestion with Streptomyces hyaluronidase (Figure 1B) or 300 kD by sonication (Figure 1D) before feeding to cells, it appeared more similar to the cell-derived hyaluronan, i.e., in a more diffuse network-like pattern throughout the cytoplasm and predominantly in the perinuclear area (compare Figures 1B and 1D with Figure 1E). This suggests that lower molecular weight hyaluronan may be more efficiently translocated to the perinuclear area and other cytoplasmic compartments. Translocation of tagged hyaluronan to the nucleus itself was not dramatic under these conditions, regardless of molecular weight. The signal within the nucleus was either weakly diffuse or faintly concentrated to the nucleoli.
Partial Lysosomal Distribution
Comparison of endogenous intracellular hyaluronan staining using a specific hyaluronan-binding probe with an antibody to the lysosomal marker LAMP-1 by confocal microscopy indicated that a substantial portion of the hyaluronan is in the lysosomal compartment in smooth muscle cells (Figure 1E). However, some hyaluronan did not colocalize with LAMP-1, such as that in the nucleolus (see below), early endosomes (data not shown) and some cytoplasmic regions near the nucleus.
Relationship of Hyaluronan with Microtubules and RHAMM
To compare the distribution of hyaluronan with cytoskeletal elements, permeabilized smooth muscle cells were double stained for endogenous hyaluronan and either f-actin or tubulin (Figure 2). The overall distribution of the hyaluronan staining within the cytoplasm most closely resembled microtubule organization (Figures 2A2C). Hyaluronan codistributed with the microtubules in the perinuclear network and also more peripherally in the cytoplasm. In contrast, little or only occasional relationship between the intracellular hyaluronan and actin filaments was apparent (Figures 2D2F). Hyaluronan-positive vesicles were frequently seen in distinct linear arrays or tracts that connected to the nuclear periphery (Figure 2G).
|
|
Using a different approach, intracellular hyaluronan-binding sites were localized by incubating fixed and permeabilized cells with fluorescein-hyaluronan and compared with the distribution of RHAMM (Figure 4). The fluorescein-hyaluronan bound directly to the microtubule structures in the perinuclear network that were stained with the anti-RHAMM antibody. This is in slight contrast to the distribution of endogenous hyaluronan and following uptake of fluorescein-hyaluronan by living cells, which localized between microtubules. Some RHAMM-positive structures, such as the thinner microtubules in the periphery of the cytoplasm, did not bind the fluorescein-hyaluronan. This suggests that some of the RHAMM associated with perinuclear microtubules is available for hyaluronan binding, i.e., not saturated. Binding of the labeled hyaluronan could be abolished by preincubation with excess unlabeled hyaluronan [data not shown, see (Evanko and Wight 1999)].
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous work has indicated that cells prefer to take up hyaluronan of low molecular weight (McGuire et al. 1987). Our results further these findings and suggest that the size of the hyaluronan may also influence its distribution intracellularly. These findings raise the question of whether the "hyaluronasome" (Stern 2003
), or membranous organelle containing hyaluronan in various states of synthesis and degradation, has some as-yet-unknown intracellular function.
Hyaluronan, Nuclear Architecture, Microtubule Function and Mitosis
A number of morphological observations suggest that there may be a relationship of intracellular hyaluronan to nuclear architecture and/or the mitotic process. These observations are as follows: (a) the presence of hyaluronan in association with heterochromatin and nucleoli (Londono and Bendayan 1988; Ripellino et al. 1989
; Kan 1990
; Evanko and Wight 1999
); (b) the binding of fluorescein-labeled hyaluronan to the nuclear periphery and to nucleoli in fixed cells with a redistribution of binding sites following serum stimulation (Evanko and Wight 1999
); (c) translocation of hyaluronan to the nucleus concurrently with stimulation of cell motility (Collis et al. 1998
); (d) the association of intracellular hyaluronan with clefts and furrows in the nucleus of some cells (Evanko and Wight 1999
); (e) elevated synthesis of hyaluronan during G2/M (Brecht et al. 1986
), and the dynamic accumulation of hyaluronan in the pericellular matrix and intracellularly in mitotic cells (Evanko and Wight 1999
; Evanko and Wight 2001
); (f) colocalization of hyaluronan with microtubules and the microtubule-associated protein, RHAMM, at the mitotic spindle, and nucleolar area (present study); and (g) positive hyaluronan staining around microtubules at the location of the cleavage furrow [current study and (Evanko and Wight 2001
)].
The abundance of a hyaluronan-positive compartment that is closely associated with the mitotic spindle and the rapid uptake that occurs at this time suggests that intracellular hyaluronan plays a role in the mitotic process. Microtubules are compression-bearing elements of the cytoskeleton that are thought to partly balance cell contraction (Stamenovic et al. 2002) and participate in key events in mitosis such as chromosome rearrangement and cleavage furrow formation. The microtubules most likely participate in the translocation of the hyaluronan to lysosomes, the nucleus and other sites. However, hyaluronan is a well-known lubricant in synovial joints and may function similarly at the cell membrane/matrix interface (Evanko et al. 1999
). This theme may also continue inside the cell. In other words, through an association with RHAMM, hyaluronan could help lubricate the intracellular motor machinery, influencing the viscosity and friction of the cellular microenvironment during microtubule-based movements (Figure 7). Alternatively, through its contribution to cytoplasmic swelling pressure, a hyaluronan-rich compartment may contribute to lateral stabilization or spacing of the microtubules and thus influence force balance within the cell during mitosis or other cell movements. In addition to a passive physical role, the hyaluronan may play an active role in modulating RHAMM function, a notion supported by an earlier study showing that blocking antibodies to RHAMM can block the binding of fluorescein-hyaluronan intracellularly (Pilarski et al. 1999
).
|
To our knowledge, this is the first report directly showing a close spatial relationship of intracellular hyaluronan with an intracellular hyaladherin and supports the idea that hyaluronan may influence the function of intracellular RHAMM (Hall et al. 1994; Collis et al. 1998
; Hofmann et al. 1998
; Pilarski et al. 1999
). RHAMM was previously found in the perinuclear microtubule network of interphase cells and in the mitotic spindle (Assmann et al. 1999
; Haddad and Turley 2000
). We also found that microtubule-associated RHAMM is available for binding to fluorescein-hyaluronan, suggesting that RHAMM is not saturated with endogenous hyaluronan, consistent with an earlier study in hematopoietic cells (Pilarski et al. 1999
). Thus, RHAMM may be an actual intracellular binding partner for the hyaluronan here. Hyaluronan interacts with phospholipid (Pasqualli-Ronchetti et al. 1997
) and is extruded through the membrane during biosynthesis. Therefore, it is not inconceivable that hyaluronan within vesicles or other membranous compartment has the capacity to interact with RHAMM. The microtubule-binding domain of RHAMM is located in the N-terminal portion (Assmann et al. 1999
) and the hyaluronan-binding sequence is in domain 5, closer to the C terminus (Yang et al. 1994
). Thus, hyaluronan conceivably could bridge two RHAMM molecules while they are associated with interphase microtubules. On the other hand, the carboxy-terminal leucine zipper targets RHAMM to the centrosome (Maxwell et al. 2003
) and overlaps with the hyaluronan-binding site, suggesting that hyaluronan could also play a competitive role in regulating mitosis. Hyaluronan staining has been previously noted to penetrate the nucleus in the prometaphase stage (Evanko and Wight 1999
), where spindle formation and chromosome alignment occur. RHAMM is known to regulate ERK kinase activity (Zhang et al. 1998
) and participates in hyaluronan-induced cell locomotion and the uptake and translocation of hyaluronan to the nucleus (Collis et al. 1998
). RHAMM and hyaluronan promote focal adhesion turnover (Hall et al. 1994
) and repeated contact of microtubules with focal adhesions promotes their disassembly (Kaverina et al. 2002
). RHAMM was recently found to interact with dynein and help maintain spindle pole stability (Maxwell et al. 2003
). Therefore, hyaluronan may regulate the effects of RHAMM on microtubule dynamics in various ways. RHAMM could mediate the binding of hyaluronan-containing vesicles to microtubules, and together they could serve in an as-yet-undefined biophysicalstructural role during the mitotic process. Alternatively, hyaluronan could regulate the amount of available RHAMM for interacting with the dynein motor complex or other signaling molecules such as ERK kinase.
It is clear that the biological activity of hyaluronan is size dependent. We found that fragmented fluorescein-labeled hyaluronan was preferentially translocated to the perinuclear area (Figure 1). In contrast, although some high molecular weight fluorescein-hyaluronan that was taken up by cells could be seen in the perinuclear area (Figure 1A) and the mitotic spindle (Figure 5G), it was mostly present in large endosomes. This extends previous studies showing that most of the intracellular hyaluronan is of low molecular weight, is present in vesicles of varying size (1001300 nm) and shape, and may be destined for eventual degradation (McGuire et al. 1987; Tammi et al. 2001
). Various mammalian hyaluronidases and hyaluronidase inhibitors that may be central to regulating hyaluronan size and catabolism have been characterized (Stern and Csoka 2000
). Given the discrete size of the hyaluronan inside cells, one can conceive of possible mechanisms whereby hyaluronan or the "hyaluronasomes" themselves could serve as novel structural/functional units. Clearly, the study of the functional and regulatory roles of intracellular hyaluronan is rapidly expanding and the testing of these and other ideas will be forthcoming. The present observations provide an additional basis for further investigation into this area.
![]() |
Acknowledgments |
---|
We would like to thank Dr Volker Assmann (Richard Dimbleby Department of Cancer Research/ICRF Laboratory, St Thomas' Hospital, London, UK) for the anti-RHAMM antibody, and Dr Robert Stern (University of San Francisco) for helpful discussions.
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aguiar DJ, Knudson W, Knudson CB (1999) Internalization of the hyaluronan receptor CD44 by chondrocytes. Exp Cell Res 252:292302[CrossRef][Medline]
Assmann V, Jenkinson D, Marshall JF, Hart IR (1999) The intracellular hyaluronan receptor RHAMM/IHABP interacts with microtubules and actin filaments. J Cell Sci 112:39433954
Brecht M, Mayer U, Schlosser E, Prehm P (1986) Increased hyaluronate synthesis is required for fibroblast detachment and mitosis. Biochem J 239:445450[Medline]
Collis L, Hall C, Lange L, Ziebel M, Prestwich R, Turley EA (1998) Rapid hyaluronan uptake is associated with enhanced motility: implications for an intracellular mode of action. FEBS Lett 440:444449[CrossRef][Medline]
Deb TB, Datta K (1996) Molecular cloning of human fibroblast hyaluronic acid-binding protein confirms its identity with P-32, a protein copurified with splicing factor SF2. J Biol Chem 271:22062212
Eggli PS, Graber W (1995) Association of hyaluronan with rat vascular endothelial cells and smooth muscle cells. J Histochem Cytochem 43:689697
Evanko SP, Angello JC, Wight TN (1999) Formation of hyaluronan and versican rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells. Arter Thromb Vasc Biol 19:10041013.
Evanko SP, Wight TN (1999) Intracellular localization of hyaluronan in proliferating cells. J Histochem Cytochem 47:13311341
Evanko SP, Wight TN (2001) Intracellular Hyaluronan. http://www.glycoforum.gr.jp/science/hyaluronan/HA20/HA20E.html Glycoforum/Science of Hyaluronan Today, http://www.glycoforum.gr.jp. V. Hascall and M. Yanagishita. Tokyo, Japan, Seikagaku, Corporation
Furukawa K, Terayama H (1977) Isolation and identification of glycosaminoglycans associated with purified nuclei from rat liver. Biochim Biophys Acta 499:278289[Medline]
Grammatikakis N, Grammatikakis A, Yoneda M, Yu Q, Banerjee SD, Toole BP (1995) A novel glycosaminoglycan-binding protein is the vertebrate homologue of the cell cycle control protein, Cdc37. J Biol Chem 270:1619816205
Haddad AA, Turley EA (2000) RHAMM protein interacts with the cytoskeleton. Mol Biol Cell 11:88a
Hall CL, Collis LA, Jing Bo A, Lange L, McNicol A, Gerrard JM, Turley EA (2001) Fibroblasts require protein kinase C activation to respond to hyaluronan with increased locomotion. Matrix Biol 20:183192[CrossRef][Medline]
Hall CL, Wang C, Lange LA, Turley EA (1994) Hyaluronan and the hyaluronan receptor RHAMM promote focal adhesion turnover and transient tyrosine kinase activity. J Cell Biol 126:575588[Abstract]
Hofmann M, Fieber C, Assmann V, Gottlicher M, Sleeman J, Plug R, Howells N, et al. (1998) Identification of IHABP, a 95 kDa intracellular hyaluronate binding protein. J Cell Sci 111:16731684
Huang L, Grammatikakis N, Yoneda M, Banerjee SD, Toole BP (2000) Molecular characterization of a novel intracellular hyaluronan-binding protein. J Biol Chem 275:2982929839
Kan FW (1990) High-resolution localization of hyaluronic acid in the golden hamster oocyte-cumulus complex by use of a hyaluronidase-gold complex. Anat Rec 228:370382[Medline]
Kaverina I, Krylyshkina O, Small JV (2002) Regulation of substrate adhesion dynamics during cell motility. Int J Biochem Cell Biol 34(7):746761[CrossRef][Medline]
Londono I, Bendayan M (1988) High-resolution cytochemistry of neuraminic acid and hexuronic acid-containing macromolecules applying the enzyme-gold approach. J Histochem Cytochem 36:10051014[Abstract]
Margolis RK, Crockett CP, Kiang W-L, Margolis RU (1976) Glycosaminoglycans and glycoproteins associated with rat brain nuclei. Biochim Biophys Acta 451:465469[Medline]
Maxwell CA, Keats JJ, Crainie M, Sun X, Yen T, Shibuya E, Hendzel M, et al. (2003) RHAMM is a centrosomal protein that interacts with dynein and maintains spindle pole stability. Mol Biol Cell 14:22622276
Maxwell CA, Pilarski LM (2000) Affinity of RHAMM isoforms for interphase and mitotic microtubules in suspension cells. Mol Biol Cell 11:200a
McGuire PG, Castellot JJ, Orkin RW (1987) Size-dependent hyaluronate degradation by cultured cells. J Cell Physiol 133:267276[Medline]
Pasqualli-Ronchetti I, Quaglino D, Mori G, Bacchelli B (1997) Hyaluronan-phospholipid interactions. J Struct Biol 120:110[CrossRef][Medline]
Pienimaki JP, Rilla K, Fulop C, Sironen RK, Karvinen S, Pasonen S, Lammi MJ, et al. (2001) Epidermal growth factor activates hyaluronan synthase 2 in epidermal keratinocytes and increases pericellular and intracellular hyaluronan. J Biol Chem 276:2042820435
Pilarski LM, Pruski E, Wizniak J, Paine D, Seeberger K, Mant MJ, Brown CB, et al. (1999) Potential role for hyaluronan and the hyaluronan receptor RHAMM in mobilization and trafficking of hematopoietic progenitor cells. Blood 93:29182927
Rieder CL, Khodjakov A (2003) Mitosis through the microscope: advances in seeing inside living cells. Science 300:9196
Ripellino JA, Margolis RU, Margolis RK (1989) Immunoelectron microscopic localization of hyaluronic acid-binding region and link protein epitopes in brain. J Cell Biol 108:18991907[Abstract]
Stamenovic D, Mijailovich SM, Tolic-Norrelykke IM, Chen J, Wang N (2002) Cell prestress. II. Contribution of microtubules. Am J Physiol Cell Physiol 282:C617C624
Stern R, Csoka AB (2000) Mammalian Hyaluronidases. http://www.glycoforum.gr.jp/science/hyaluronan/HA15/HA15E.html Glycoforum/Science of Hyaluronan Today, http://www.glycoforum.gr.jp. V. Hascall and M. Yanagashita. Tokyo, Japan, Seikagaku, Corporation
Stern R (2003) Devising a pathway for hyaluronan catabolism: are we there yet? Glycobiology 13:105R115R
Tammi R, Rilla K, Pienimaki J-P, MacCallum DK, Luukonen M, Hascall VC, Tammi M (2001) Hyaluronan enters keratinocytes by a novel endocytic route for catabolism. J Biol Chem (submitted)
Tammi R, Tammi M (1991) Correlations between hyaluronan and epidermal proliferation as studied by [3H]glucosamine and [3H]thymidine incorporations and staining of hyaluronan on mitotic keratinocytes. Exp Cell Res 195:524527[CrossRef][Medline]
Turley EA, Torrance J (1984) Localization of hyaluronate and hyaluronate-binding protein on motile and non-motile fibroblasts. Exp Cell Res 161:1728
Underhill CB, Toole BP (1979) Binding of hyaluronate to the surface of cultured cells. J Cell Biol 82:475484[Abstract]
Yang B, Yang BL, Savani RC, Turley EA (1994) Identification of a common hyaluronan binding motif in the hyaluronan binding proteins RHAMM, CD44 and link protein. EMBO J 13:286296[Abstract]
Zhang S, Chang MCY, Zylka D, Turley S, Harrison R, Turley EA (1998) The hyaluronan receptor RHAMM regulates extracellular-regulated kinase. J Biol Chem 273:1134211348
Zhou R, Wu X, Skalli O (2002) The hyaluronan receptor RHAMM/IHABP in astrocytoma cells: expression of a tumor-specific variant and association with microtubules. J Neurooncol 59:1526[CrossRef][Medline]