Receptor for hyaluronan mediated motility (RHAMM/HMMR) is a novel target for promoting subcutaneous adipogenesis.

Hyaluronan, CD44 and the Receptor for Hyaluronan-Mediated Motility (RHAMM, gene name HMMR) regulate stem cell differentiation including mesenchymal progenitor differentiation. Here, we show that CD44 expression is required for subcutaneous adipogenesis, whereas RHAMM expression suppresses this process. We designed RHAMM function blocking peptides to promote subcutaneous adipogenesis as a clinical and tissue engineering tool. Adipogenic RHAMM peptides were identified by screening for their ability to promote adipogenesis in culture assays using rat bone marrow mesenchymal stem cells, mouse pre-adipocyte cell lines and primary human subcutaneous pre-adipocytes. Oil red O uptake into fat droplets and adiponectin production were used as biomarkers of adipogenesis. Positive peptides were formulated in either collagen I or hyaluronan (Orthovisc) gels then assessed for their adipogenic potential in vivo following injection into dorsal rat skin and mammary fat pads. Fat content was quantified and characterized using micro CT imaging, morphometry, histology, RT-PCR and ELISA analyses of adipogenic gene expression. Injection of screened peptides increased dorsal back subcutaneous fat pad area (208.3 ± 10.4 mm2versus control 84.11 ± 4.2 mm2; p < 0.05) and mammary fat pad size (45 ± 11 mg above control background, p = 0.002) in female rats. This effect lasted >5 weeks as detected by micro CT imaging and perilipin 1 mRNA expression. RHAMM expression suppresses while blocking peptides promote expression of PPARγ, C/EBP and their target genes. Blocking RHAMM function by peptide injection or topical application is a novel and minimally invasive method for potentially promoting subcutaneous adipogenesis in lipodystrophic diseases and a complementary tool to subcutaneous fat augmentation techniques.

Hyaluronan metabolism in remodeling extracellular matrix: probes for imaging and therapy of breast cancer.

Clinical and experimental evidence increasingly support the concept of cancer as a disease that emulates a component of wound healing, in particular abnormal stromal extracellular matrix remodeling. Here we review the biology and function of one remodeling process, hyaluronan (HA) metabolism, which is essential for wound resolution but closely linked to breast cancer (BCA) progression. Components of the HA metabolic cycle (HAS2, SPAM1 and HA receptors CD44, RHAMM/HMMR and TLR2) are discussed in terms of their known functions in wound healing and in breast cancer progression. Finally, we discuss recent advances in the use of HA-based platforms for developing nanoprobes to image areas of active HA metabolism and for therapeutics in breast cancer.

Peptides that mimic glycosaminoglycans: high-affinity ligands for a hyaluronan binding domain.

BACKGROUND: Hyaluronan (HA) is a non-sulfated glycosaminoglycan (GAG) that promotes motility, adhesion, and proliferation in mammalian cells, as mediated by cell-surface HA receptors. We sought to identify non-carbohydrate ligands that would bind to and activate cell-surface HA receptors. Such analogs could have important therapeutic uses in the treatment of cancer, wound healing, and arthritis, since such ligands would be resistant to degradation by hyaluronidase (HAse). RESULTS: Peptide ligands that bind specifically to the recombinant HA binding domain (BD) of the receptor for hyaluronan-mediated motility (RHAMM) were obtained by screening two peptide libraries: (i) random 8-mers and (ii) biased 8-mers with alternating acidic side chains, i.e. XZXZXZXZ (X=all-L-amino acids except Cys, Lys, or Arg; Z=D-Asp, L-Asp, D-Glu, or L-Glu). Selectivity of the peptide ligands for the HABD was established by (i) detection of binding of biotin- or fluorescein-labeled peptides to immobilized proteins and (ii) fluorescence polarization of FITC-labeled peptides with the HABD in solution. HA competitively displaced binding of peptides to the HABD, while other GAGs were less effective competitors. The stereochemistry of four biased octapeptides was established by synthesis of the 16 stereoisomers of each peptide. Binding assays demonstrated a strong preference for alternating D and L configurations for the acidic residues, consistent with the calculated orientation of glucuronic acid moieties of HA. CONCLUSIONS: Two classes of HAse-resistant peptide mimetics of HA were identified with high affinity, HA-compatible binding to the RHAMM HABD. This demonstrated that non-HA ligands specific to a given HA binding protein could be engineered, permitting receptor-specific targeting.

Active erk regulates microtubule stability in H-ras-transformed cells.

Increasing evidence suggests that activated erk regulates cell functions, at least in part, by mechanisms that do not require gene transcription. Here we show that the map kinase, erk, decorates microtubules (MTs) and mitotic spindles in both parental and mutant active ras-transfected 10T1/2 fibroblasts and MCF10A breast epithelial cells. Approximately 20% of total cellular erk decorated MTs in both cell lines. A greater proportion of activated erk was associated with MTs in the presence of mutant active H-ras than in parental cells. Activation of erk by the ras pathway coincided with a decrease in the stability of MT, as detected by a stability marker. The MKK1 inhibitor, PD98059 and transfection of a dominant negative MKK1 blocked ras-induced instability of MTs but did not modify the association of erk with MTs or affect MT stability of the parental cells. These results indicate that the subset of active erk kinase that associates with MTs contributes to their instability in the presence of a mutant active ras. The MT-associated subset of active erk likely contributes to the enhanced invasive and proliferative abilities of cells containing mutant active H-ras.

Identification of sequence, protein isoforms, and distribution of the hyaluronan-binding protein RHAMM in adult and developing rat brain.

The protein RHAMM (for "receptor for hyaluronan-mediated motility"; CD168) is a member of the hyaladherin family of hyaluronan-binding proteins. RHAMM has a role in cell signaling, migration, and adhesion via interactions with hyaluronan, microtubules, actin, calmodulin, and components of the extracellular regulated kinase (erk) signaling pathway. Based on previous findings of potentially similar roles in neural cells in culture, we investigated the molecular characteristics, protein expression profile, and distribution of RHAMM in rat brain. Reverse transcriptase-polymerase chain reaction (RT-PCR) using RNA isolated from adult rat brain yielded a single RHAMM sequence of 2.1 kilobases encoding a protein of 82.4 kDa. RHAMM is subject to alternate splicing in other systems, but no RT-PCR evidence was found for splice variants in brain, although our analysis does not rule out this possibility. The amino acid sequence displayed homology with human and murine RHAMM (74% and 80%, respectively) but contained only one copy of a 21-amino-acid sequence that is repeated five times in the murine homologue. By using anti-RHAMM antibodies, several RHAMM isoforms were identified in brain. Immunohistochemically, RHAMM was found in the vast majority of neurons and in many oligodendrocytes throughout brain, with heterogeneous levels among cell populations, and was confined to the somata and initial processes of these cells. RHAMM was detected in neurons of cerebral cortex and most subcortical and brainstem structures at postnatal day 1 and exhibited an adult distribution pattern by postnatal day 5. High levels were detected in oligodendrocytes by postnatal day 10. The widespread expression of RHAMM in adult and developing brain implies a role for this protein and its ligand hyaluronan in key events of cell signaling and cytoskeletal regulation in the CNS.

Subcellular distribution, calmodulin interaction, and mitochondrial association of the hyaluronan-binding protein RHAMM in rat brain.

The CNS contains high levels of the glycosaminoglycan hyaluronan, and neural cells express a variety of proteins that are members of the hyaladherin family of hyaluronan-binding proteins. We have previously shown that the hyaladherin RHAMM (receptor for hyaluronan-mediated motility; CD168) is expressed by neural cells in culture; plays a role in astrocyte motility, neurite migration, and axonal growth; and is widely distributed in neurons and oligodendrocytes of developing and adult rat CNS. Here we demonstrate differential localization of various forms of RHAMM in subcellular fractions of adult rat brain. Western blotting indicated the presence of 66, 75, and 85-90 kDa molecular weight RHAMM forms in whole-brain homogenates. Subfractionation revealed enrichment of the 66 and 85-90 kDa forms in soluble fractions, whereas the 75 kDa form was enriched in mitochondrial fractions. This latter form was retained in osmotically shocked mitochondria, but was liberated by alkali carbonate, suggesting a nonintrinsic mitochondrial membrane association. By double immunohistochemical labeling for RHAMM and the mitochondrial marker cytochrome oxidase, RHAMM was localized to isolated mitochondria in vitro and to neuronal mitochondria in vivo. Hyaluronan-sepharose chromatography and cetylpiridinium chloride precipitation confirmed the hyaluronan-binding capacity of RHAMM forms. By calmodulin-affinity chromatography, endogenously expressed brain RHAMM was demonstrated to bind calmodulin in a Ca2+-dependent manner. These results, together with reports of RHAMM association with actin and microtubules in other systems, suggest a role of RHAMM in calmodulin-mediated cell signaling to cytoskeletal elements and/or mitochondria in the CNS and invoke novel functions of its interactions with hyaluronan.

Fibroblasts require protein kinase C activation to respond to hyaluronan with increased locomotion.

Hyaluronan (HA) stimulates the motility of some but not all cell types. Here, we show that HA-promoted random motility of ras-transformed 10T1/2 (C3) fibroblasts requires activation of protein kinase C and is associated with rapid uptake of HA in a CD44 and RHAMM-dependent manner. The addition of HA to parental 10T1/2 fibroblasts (parental cells) does not stimulate random motility, but these cells can be 'primed' to respond to HA by treatment with the phorbol ester, PMA, for 4-6 h. This effect of PMA requires protein synthesis, PKC activity and is associated with enhanced uptake of HA. These results suggest that the ability of cells to respond to HA is regulated by a protein kinase C-dependent process that may promote uptake of HA.

The hyaluronan receptor RHAMM regulates extracellular-regulated kinase.

We have identified two RHAMM (receptor for hyaluronan-mediated motility) isoforms that encode an alternatively spliced exon 4 (Hall, C. L., Yang, B., Yang, X., Zhang, S., Turley, M., Samuel, S., Lange, L. A., Wang, C., Curpen, G. D., Savani, R. C., Greenberg, A. H., and Turley, E. A. (1995) Cell 82, 19-26 and Wang, C., Entwistle, J., Hou, G., Li, Q., and Turley, E. A. (1996) Gene 174, 299-306). One of these, RHAMM variant 4 (RHAMMv4), is transforming when overexpressed and regulates Ras signaling (Hall et al.). Here we note using flow cytometry and confocal analysis that RHAMM isoforms encoding exon 4 occur both on the cell surface and in the cytoplasm. Epitope-tagging experiments indicate that RHAMMv4 occurs only in the cytoplasm. Several observations suggest that both cell surface RHAMM isoforms and RHAMMv4 are involved in regulating extracellular-regulated kinase (ERK) activity. Affinity-purified anti-RHAMM exon 4 antibodies block the ability of platelet-derived growth factor to activate ERK, and these reagents modify the protein tyrosine phosphorylation profile of proteins resulting from treatment with platelet-derived growth factor. A dominant negative form of RHAMMv4 inhibits mutant active Ras activation of ERK and coimmunoprecipitates with both mitogen-activated protein kinase kinase and ERK, suggesting that the intracellular RHAMMv4 acts downstream of Ras, possibly at the level of mitogen-activated protein kinase kinase-ERK interactions. Consistent with this, overexpression of RHAMMv4 constitutively activates ERK. These results identify a novel mechanism for the regulation of the Ras-ERK signaling pathway and suggest that RHAMM plays multiple roles in this regulation.

The overexpression of RHAMM, a hyaluronan-binding protein that regulates ras signaling, correlates with overexpression of mitogen-activated protein kinase and is a significant parameter in breast cancer progression.

RHAMM is an oncogene that regulates signaling through ras and controls mitogen-activated protein kinase [extracellular signal-regulated protein kinase (ERK)] expression in embryonic murine fibroblasts. ERK is a dual-specificity kinase that controls expression of proteins relevant to tumorigenesis, proliferation, and motility. To assess whether RHAMM and ERK are involved in human breast tumor progression, we examined RHAMM, ras, and ERK expression in two cohorts of breast cancer patients using reverse transcription-PCR and immunocytochemistry. We show that overexpression of RHAMM in primary tumors of two patient cohorts was significantly prognostic of poor outcome in breast cancer progression. Furthermore, RHAMM overexpression occurred within subsets of tumor cells in the primary tumor, and this staining pattern was associated with lymph node metastases. The metastases exhibited a significantly higher level of staining for RHAMM than did the primary tumor. RHAMM expression strongly correlated with overexpression of both ras and ERK, although overexpression of either of these two signaling molecules was not by itself a prognostic indicator. These results identify a new parameter that is involved in lymph node metastasis of primary breast cancers and suggest that quantification of RHAMM overexpression may be a useful prognostic indicator for breast carcinoma progression.

Rapid hyaluronan uptake is associated with enhanced motility: implications for an intracellular mode of action.

Texas red-labeled hyaluronan (TR-HA) is rapidly taken up in a CD44 independent manner into ras-transformed 10T1/2 fibroblasts, where it accumulates in both cell ruffles/lamellae, the perinuclear area, and the nucleus. HA does not accumulate in the cell ruffles/lamellae of parental 10T1/2 cells. Addition of HA to ras-transformed cells promotes their random motility but has no effect on 10T1/2 cell motility. 10T1/2 cells can be modified to take up HA into cell ruffles by exposure to phorbol ester or direct microinjection of HA into cells. Both treatments significantly stimulate 10T1/2 cell motility.

pp60(c-src) is required for cell locomotion regulated by the hyaluronanreceptor RHAMM.

The tyrosine kinase pp60(c-src) has been implicated as a regulator of focal adhesion formation and cell spreading. Here we show that c-src also regulates cell motility and is a key component in the signaling pathway triggered by the motogenic hyaluronan receptor RHAMM, which has been shown to regulate focal adhesion turnover and to regulate ras. Fibroblasts derived from mice lacking src, (src (-/-)), have a random locomotion rate that is significantly slower than the corresponding wild-type fibroblasts. Cell locomotion in these mutant cells is restored by the expression of c-src containing a functional kinase domain, but not by the expression of a kinase-deficient src or by a truncated src containing only functional SH2 and SH3 domains. RHAMM is also required for the restoration of src (-/-) cell locomotion. Thus, the motility of cells expressing c-src is reduced to src (-/-) levels by anti-RHAMM blocking antibodies while the cell locomotion of src (-/-) fibroblasts remains unaffected by anti-RHAMM antibodies. We predict that src acts downstream of RHAMM in the regulation of motility, since the expression of a dominant negative src significantly inhibits RHAMM-dependent ras and serum regulated cell locomotion, the expression of v-src enhances cell motility in a RHAMM independent fashion, and there is a physical and functional assocation between src and RHAMM in ras-transformed cells. However, we suggest that RHAMM regulates focal adhesion turnover via additional src-independent mechanisms. Thus, v-src is unable to turnover focal adhesions in the absence of RHAMM. These results directly demonstrate for the first time a role for src in the regulation of cell locomotion and confirm a key and complex role for src in the regulation of the actin cycle.

The characterization of a human RHAMM cDNA: conservation of the hyaluronan-binding domains.

A full-length human RHAMM cDNA clone was isolated by a combination of screening a human breast cDNA expression library with the murine RHAMM 2 cDNA as well as 5' RACE and RT-PCR using messenger RNA from human breast cell line (MCF-10A). The full-length cDNA contained 725 aa that encoded an 84 kDa protein. Although the coding region of the human RHAMM cDNA resembles the murine RHAMM v4, it has additional unique N-terminal (489 bp) and C-terminal (33 bp) regions. Also, only 1 of 5 repeat sequences encoded in the murine cDNA are present in human cDNA. The overall homology between the overlapping region of human and mouse RHAMM v4 cDNA clone is 85%, but the HA binding motif (B[X7]B), shown to be critical for the signaling capability of this receptor, is 100% conserved.

HA receptors: regulators of signalling to the cytoskeleton.

Hyaluronan (HA) is a ubiquitous component of the extracellular matrix (ECM) and occurs transiently in both the cell nucleus and cytoplasm. It has been shown to promote cell motility, adhesion, and proliferation and thus it has an important role in such processes as morphogenesis, wound repair, inflammation, and metastasis. These processes require massive cell movement and tissue reorganization and are always accompanied by elevated levels of HA. Many of the effects of HA are mediated through cell surface receptors, three of which have been molecularly characterized, namely CD44, RHAMM, and ICAM-1. Binding of the HA ligand to its receptors triggers signal transduction events which, in concert with other ECM and cytoskeletal components, can direct cell trafficking during physiological and pathological events. The HA mediated signals are transmitted, at least in part, by the activation of protein phosphorylation cascades, cytokine release, and the stimulation of cell cycle proteins. A variety of extracellular signals regulate the expression of both HA and the receptors necessitating that HA-receptor signalling is a tightly controlled process. Regulated production of soluble forms of the receptors, alternately spliced cell surface isoforms, and glycosylation variants of these receptors can dramatically modulate HA binding, ligand specificity, and stimulation of the signalling pathway. When these processes are deregulated cell behaviour becomes uncontrolled leading to developmental abnormalities, abnormal physiological responses, and tumorigenesis. The elucidation of the molecular mechanisms regulating HA-mediated events will not only contribute greatly to our understanding of a variety of disease processes but will also offer many new avenues of therapeutic intervention.

Increased connexin-43 and gap junctional communication correlate with altered phenotypic characteristics of cells overexpressing the receptor for hyaluronic acid-mediated motility.

Gap junctional intercellular communication (GJIC) and connexin expression are often altered during cell migration, growth, and transformation, each of which is accompanied by cytoskeletal reorganization. Recently, transfection of fibroblast cells with various isoforms of the receptor for hyaluronic acid-mediated motility (RHAMM) was shown to have profound and differential effects on motility, growth, and cell contact behavior as well as on elements of the actin-containing cytoskeleton. These cells thus provide an ideal system in which to investigate parameters implicated in regulation of GJIC as well as expression of connexin-43 (Cx43) in fibroblasts. We used 10T1/2 fibroblast cell lines transfected with RHAMM isoforms or a dominant negative mutated form of RHAMM that blocks the function of endogenous RHAMM. Increased RHAMM expression in the various cell lines was correlated with increased Cx43 and GJIC. These changes were accompanied by a loss of contact inhibition and decreased focal adhesions in all, and elevated motility of most but not all, cell lines tested. RHAMM-induced transformation also resulted in elevated GJIC and Cx43 levels. Reversion to normal growth, motility, and focal adhesion density following transfection of H-ras-transformed fibroblasts with the mutant form of RHAMM was associated with decreases in both Cx43 expression and GJIC. Transfection of 10T1/2 fibroblasts with RHAMM II (exons 5-14) produced altered contact behavior and increased both Cx43 and GJIC but had no effect on motility. All cells expressing high levels of RHAMM, regardless of the isoform, exhibited a lower density of focal adhesions, which corresponds to a reduced organizational state of the cytoskeleton. These results indicate that regulation of GJIC most strongly correlates with altered focal adhesion and cytoskeleton organization that can lead to various secondary responses, including motility, growth, and transformation, and suggest that RHAMM regulates GJIC and Cx43 expression possibly through its actions on focal adhesions and the associated cytoskeleton.

Transforming growth factor-beta1 stimulates multiple protein interactions at a unique cis-element in the 3'-untranslated region of the hyaluronan receptor RHAMM mRNA.

The receptor for hyaluronan mediated motility (RHAMM) gene expression is markedly elevated in fibrosarcomas exposed to transforming growth factor-beta1 (TGF-beta1). The half-life of RHAMM mRNA was increased by 3 fold in cells treated with TGF-beta1, indicating that growth factor regulation of RHAMM gene expression at least in part involves a posttranscriptional mechanism. Our studies demonstrated that a unique 30-nucleotide (nt) region that has three copies of the sequence, GCUUGC, was the TGF-beta1-responsive region in the 3'-untranslated region (3'-UTR) that mediated message stability. This region interacted specifically with cytoplasmic trans-factors to form multiple protein complexes of approximately 175, 97, 63, 26, and 17 kDa post-TGF-beta1 treatment, suggesting a role for these complexes in the mechanism of action of TGF-beta1-induced message stabilization. Insertion of the 3'-UTR into the chloramphenicol acetyltransferase gene conferred TGF-beta1 induced stability of chloramphenicol acetyltransferase-hybrid RNA in stably transfected cells, while the same insert carrying a deletion containing the 30-nt region had no significant effect on mRNA stability. These results provide a model of RHAMM message regulation in which TGF-beta1-mediated alteration of RHAMM message stability involves the up-regulation of multiple protein interactions with a 30-nt cis-element stability determinant in the 3'-UTR. This model also suggests that this 30-nt base region functions in cis to destabilize RHAMM mRNA in resting normal cells.

Soluble hyaluronan receptor RHAMM induces mitotic arrest by suppressing Cdc2 and cyclin B1 expression.

The hyaluronan (HA) receptor RHAMM is an important regulator of cell growth. Overexpression of RHAMM is transforming and is required for H-ras transformation. The molecular mechanism underlying growth control by RHAMM and other extracellular matrix receptors remains largely unknown. We report that soluble RHAMM induces G2/M arrest by suppressing the expression of Cdc2/Cyclin B1, a protein kinase complex essential for mitosis. Down-regulation of RHAMM by use of dominant negative mutants or antisense of mRNA also decreases Cdc2 protein levels. Suppression of Cdc2 occurs as a result of an increased rate of cdc2 mRNA degradation. Moreover, tumor cells treated with soluble RHAMM are unable to form lung metastases. Thus, we show that mitosis is directly linked to RHAMM through control of Cdc2 and Cyclin B1 expression. Failure to sustain levels of Cdc2 and Cyclin B1 proteins leads to cell cycle arrest.

Hyaluronan-dependent motility of B cells and leukemic plasma cells in blood, but not of bone marrow plasma cells, in multiple myeloma: alternate use of receptor for hyaluronan-mediated motility (RHAMM) and CD44.

We investigated the ability of blood B cells, bone marrow (BM) plasma cells, and terminal leukemic plasma cells (T-PCL) from patients with multiple myeloma (MM) to migrate on extracellular matrix proteins. Hyaluronan (HA), but not collagen type I, collagen type IV, or laminin, promoted migration of MM blood B cells, as determined by time-lapse video microscopy. Between 13% and 20% of MM blood B cells migrated on HA with an average velocity of 19 micron/min, and greater than 75% of MM blood B cells exhibited vigorous cell movement and plasma membrane deformation, as did circulating T-PCL and extraskeletal plasma cells from patients with MM. In contrast, plasma cells obtained from BM of patients with MM lacked motility on all substrates tested and did not exhibit cell membrane protrusions or cellular deformation. MM blood B cells and MM plasma cells from all sources examined expressed the HA-binding receptors receptor for HA-mediated motility (RHAMM) and CD44. On circulating MM B cells, both RHAMM and CD44 participated in HA-binding, indicating their expression ex vivo in an activated conformation. In contrast, for the majority of BM plasma cells in the majority of patients with MM, expression of RHAMM or CD44 was not accompanied by HA binding. A minority of patients did have HA-binding BM plasma cells, involving both RHAMM and CD44, as evidenced by partial blocking with monoclonal antibodies (MoAbs) to RHAMM or to CD44. Despite HA binding by both RHAMM and CD44, migration of MM blood B cells on HA was inhibited by anti-RHAMM but not by anti-CD44 MoAbs, indicating that RHAMM but not CD44 mediates motility on HA. Thus, circulating B and plasma cells in MM exhibit RHAMM- and HA-dependent motile behavior indicative of migratory potential, while BM plasma cells are sessile. We speculate that a subset(s) of circulating B or plasma cells mediates malignant spread in myeloma.