Inducible hyaluronan production reveals differential effects on prostate tumor cell growth and tumor angiogenesis

Prostate cancer progression can be predicted in human tumor biopsies by abundant hyaluronan (HA) and its processing enzyme, the hyaluronidase HYAL1. Accumulation of HA is dictated by the balance between expression levels of HA synthases, the enzymes that produce HA polymers, and hyaluronidases, which process polymers to oligosaccharides. Aggressive prostate tumor cells express 20-fold higher levels of the hyaluronan synthase HAS3, but the mechanistic relevance of this correlation has not been determined. We stably overexpressed HAS3 in prostate tumor cells. Adhesion to extracellular matrix and cellular growth kinetics in vitro were significantly reduced. Slow growth in culture was restored either by exogenous addition of hyaluronidase or by stable HYAL1 coexpression. Coexpression did not improve comparably slow growth in mice, however, suggesting that excess hyaluronan production by HAS3 may alter the balance required for induced tumor growth. To address this, we used a tetracycline-inducible HAS3 expression system in which hyaluronan production could be experimentally controlled. Adjusting temporal parameters of hyaluronan production directly affected growth rate of the cells. Relief from growth suppression in vitro but not in vivo by enzymatic removal of HA effectively uncoupled the respective roles of hyaluronan in growth and angiogenesis, suggesting that growth mediation is less critical to establishment of the tumor than early vascular development. Collectively results also imply that HA processing by elevated HYAL1 expression in invasive prostate cancer is a requirement for progression.     

Hyaluronan synthases and hyaluronidases in the kidney during changes in hydration status

Hyaluronan is a large glycosaminoglycan that is abundant in the interstitium of the renal medulla/papilla. Papillary hyaluronan increases during hydration and decreases during dehydration. Due to its gel properties and ability to retain large volumes of water, hyaluronan plays a role in renal water handling by affecting the permeability characteristics of the papillary interstitium. The focus of the present investigation was the regulation of hyaluronan metabolism in the kidney, especially during variations in hydration status.In control papillas, HAS 2 mRNA was heavily expressed and HAS 1 and 3 mRNA were weakly distributed. HYALs 1–3 mRNA were found at high expression and HYAL 4 was only weakly expressed. In hydrated animals, the diuretic response (12-fold) was followed by a 58% elevation in papillary hyaluronan and a 45% reduction in the excreted urinary hyaluronidase activity. No difference was determined in HAS 1–3 mRNA or HYAL 1, 3–4 mRNA expression, suggesting a change in activity rather than amount of protein. In dehydrated animals, antidiuresis was followed by a 22% reduction in papillary hyaluronan and a 62% elevation in excreted urinary hyaluronidase activity. Plasma vasopressin was 2.8-fold higher in dehydrated vs. hydrated rats.In conclusion, HAS 2 appears a major contributor to the baseline levels of hyaluronan. Reduced HAS 2 gene expression and increased excreted urinary hyaluronidase activity during dehydration contribute to the reduced amount of hyaluronan and to antidiuretic response.     

The distribution of renal hyaluronan and the expression of hyaluronan synthases during water deprivation in the Spinifex hopping mouse, Notomys alexis

Hyaluronan (HA) is a glycosaminoglycan that is synthesized by a family of enzymes called hyaluronan synthases (HASs), of which there are three isoforms (HAS1, 2 and 3) in mammals. The HASs have different tissue expression patterns and function, indicating that synthesis of HA and formation of the HA matrix may be regulated by various factors. The HA matrix has an important role in renal water handling and the production of a concentrated urine. We investigated the distribution of HA and the expression of HAS1, HAS2 and HAS3 mRNAs in the kidney of the Spinifex hopping mouse, Notomys alexis, a native Australian desert rodent that is reported to produce the most concentrated urine of any mammal. After periods of three, seven and fourteen days of water deprivation, the distribution of renal HA changed considerably, and there was a general down-regulation of HAS mRNA expression. It is proposed that the regulation of HA synthesis by the different HAS isoforms during water deprivation in N. alexis, could be influenced by the molecular mass of the HA chains produced by each isoform, followed by the rate at which the individual HAS produces HA.     

The distribution of renal hyaluronan and the expression of hyaluronan synthases during water deprivation in the Spinifex hopping mouse, Notomys alexis.

Hyaluronan (HA) is a glycosaminoglycan that is synthesized by a family of enzymes called hyaluronan synthases (HASs), of which there are three isoforms (HAS1, 2 and 3) in mammals. The HASs have different tissue expression patterns and function, indicating that synthesis of HA and formation of the HA matrix may be regulated by various factors. The HA matrix has an important role in renal water handling and the production of a concentrated urine. We investigated the distribution of HA and the expression of HAS1, HAS2 and HAS3 mRNAs in the kidney of the Spinifex hopping mouse, Notomys alexis, a native Australian desert rodent that is reported to produce the most concentrated urine of any mammal. After periods of three, seven and fourteen days of water deprivation, the distribution of renal HA changed considerably, and there was a general down-regulation of HAS mRNA expression. It is proposed that the regulation of HA synthesis by the different HAS isoforms during water deprivation in N. alexis, could be influenced by the molecular mass of the HA chains produced by each isoform, followed by the rate at which the individual HAS produces HA.     

Ability of hyaluronidase 2 to degrade extracellular hyaluronan is not required for its function as a receptor for jaagsiekte sheep retrovirus

Jaagsiekte sheep retrovirus (JSRV) uses hyaluronidase 2 (Hyal2) as a cell entry receptor. By making inactivating mutations to the catalytic residues of human Hyal2, we found that hyaluronidase activity was dispensable for its receptor function. The affinities of the JSRV envelope glycoprotein for Hyal2 and the Hyal2 mutant were similar, and hyaluronan did not block either high-affinity interaction or virus infection. While generating the Hyal2 mutant, we discovered that our previous analysis of the hyaluronidase activity of Hyal2 was affected by a contaminating hyaluronan lyase, which we have identified as the occlusion-derived baculovirus E66 protein of the recombinant baculovirus used to produce Hyal2. We now report that purified human Hyal2 is a weak acid-active hyaluronidase.     

Renal interstitial hyaluronan: functional aspects during normal and pathological conditions

The glycosaminoglycan (GAG) hyaluronan (HA) is recognized as an important structural component of the extracellular matrix, but it also interacts with cells during embryonic development, wound healing, inflammation, and cancer; i.e., important features in normal and pathological conditions. The specific physicochemical properties of HA enable a unique hydration capacity, and in the last decade it was revealed that in the interstitium of the renal medulla, where the HA content is very high, it changes rapidly depending on the body hydration status while the HA content of the cortex remains unchanged at very low amounts. The kidney, which regulates fluid balance, uses HA dynamically for the regulation of whole body fluid homeostasis. Renomedullary HA elevation occurs in response to hydration and during dehydration the opposite occurs. The HA-induced alterations in the physicochemical characteristics of the interstitial space affects fluid flux; i.e., reabsorption. Antidiuretic hormone, nitric oxide, angiotensin II, and prostaglandins are classical hormones/compounds involved in renal fluid handling and are important regulators of HA turnover during variations in hydration status. One major producer of HA in the kidney is the renomedullary interstitial cell, which displays receptors and/or synthesis enzymes for the hormones mentioned above. During several kidney disease states, such as ischemia-reperfusion injury, tubulointerstitial inflammation, renal transplant rejection, diabetes, and kidney stone formation, HA is upregulated, which contributes to an abnormal phenotype. In these situations, cytokines and other growth factors are important stimulators. The immunosuppressant agent cyclosporine A is nephrotoxic and induces HA accumulation, which could be involved in graft rejection and edema formation. The use of hyaluronidase to reduce pathologically overexpressed levels of tissue HA is a potential therapeutic tool since diuretics are less efficient in removing water bound to HA in the interstitium. Although the majority of data describing the role of HA originate from animal and cell studies, the available data from humans demonstrate that an upregulation of HA also occurs in diabetic kidneys, in transplant-rejected kidneys, and during acute tubular necrosis. This review summarizes the current knowledge regarding interstitial HA in the role of regulating kidney function during normal and pathological conditions. It encompasses mechanistic insights into the background of the heterogeneous intrarenal distribution of HA; i.e., late nephrogenesis, its regulation during variations in hydration status, and its involvement during several pathological conditions. Changes in hyaluronan synthases, hyaluronidases, and binding receptor expression are discussed in parallel.     

Domain motions of hyaluronan lyase underlying processive hyaluronan translocation

Hyaluronan lyase (Hyal) is a surface enzyme occurring in many bacterial organisms including members of Streptococcus species. Streptococcal Hyal primarily degrades hyaluronan‐substrate (HA) of the extracellular matrix. This degradation appears to facilitate the spread of this bacterium throughout host tissues. Unlike purely endolytic degradation of its other substrates, unsulfated chondroitin or some chondroitin sulfates, the degradation of HA by Hyal proceeds by processive exolytic cleavage of one disaccharide at a time following an initial endolytic cut. Molecular dynamics (MD) studies of Hyal from Streptococcus pneumoniae are presented that address the enzyme's molecular mechanism of action and the role of domain motions for processive functionality. The analysis of extensive sub‐microsecond MD simulations of this enzyme action on HA‐substrates of different lengths and the connection between the domain dynamics of Hyal and the translocation of the HA‐substrate reveals that opening/closing and twisting domain motions of the Hyal are intimately linked to processive HA degradation. Enforced simulations confirmed this finding as the domain motions in SpnHyal were found to be induced by enforced substrate translocation. These results establish the dynamic interplay between Hyal flexibility and substrate translocation and provide insight into the processive mechanism of Hyal. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Mammalian hyaluronan synthases

Three mammalian hyaluronan (HA) synthase genes, HAS1, HAS2, and HAS3, have been cloned and expressed, allowing the mechanisms for regulation of HA biosynthesis and function to be studied. The hyaluronan synthase (HAS) isoforms differ in kinetic characteristics and product size. The expression of each HAS isoform is controlled in a different fashion when mammalian cells are stimulated by various cytokines and the expression patterns are both spatially and temporally regulated during embryonic development. The existence of three different HAS isoforms with different characteristics implies that the broad range of biological and physiological roles performed by HA are regulated by controlling the activities and expression of the HAS isoforms. This review focuses on recent findings on the regulatory mechanisms for controlling HA biosynthesis and provides new insights into the enzymic basis for the functional regulation of HA.     

Cervical expression of hyaluronan synthases varies with the stage of the estrous cycle in the ewe

The natural cervical relaxation which occurs at estrus in the ewe may be initiated by binding of hyaluronan (HA) to its receptor CD44. Indeed, we have previously shown that HA content and fragment size in the ovine cervix varies with the stage of the estrous cycle. Despite the importance of cervical relaxation in promoting sperm transport and facilitating the possible development of transcervical artificial insemination (AI), the mechanisms coordinating these changes in HA content remain to be defined. Hyaluronan synthases (HAS) 1, 2, and 3 regulate HA biosynthesis and herein, we describe the changing pattern of HAS isoform expression during the estrous cycle to determine whether this may underpin HA-mediated changes in relaxation of the ovine cervix. Accordingly, cervices were collected from 24 cyclic sheep (n = 8 / group) at the luteal, pre-luteinizing hormone (LH) and post-LH surge stages. Protein and mRNA expression for HAS 1, 2 and 3 was determined in five different tissue layers (epithelium, subepithelial stroma, and longitudinal, circular and transverse muscle) of the vaginal, mid and uterine regions of each cervix by immunohistochemistry and in situ hybridization, respectively. HA synthases were expressed in all the tissue layers and regions of the cervix, and the pattern of expression was similar for mRNA and protein. HAS1 protein and mRNA expression was significantly (P ≤ 0.05) higher at the pre-LH surge stage, while HAS 2 and 3 protein and mRNA expression was significantly (P ≤ 0.001) higher at the luteal stage. Overall, both HAS protein and mRNA expression was significantly (P ≤ 0.001) higher in the epithelial layer and the vaginal region. These findings are in accordance with our previous results and explain the differences observed in the HA content and differing HA fragment size at different stages of the estrous cycle.     

Parallel up-regulation of FGF-2 and hyaluronan during development of cardiac hypertrophy in rat

The importance of glycosaminoglycan hyaluronan (HA) and its receptor CD44 in cell proliferation is becoming increasingly evident. Expression of the genes coding for hyaluronan synthase 1 (HAS1), HAS2, HAS3, CD44, fibroblast growth factor-2 (FGF-2), and FGF receptor-1 (FGFR-1) and the histological evidence for increases of HA and CD44 were investigated in an experimental rat model of cardiac hypertrophy. The abdominal aorta was ligated to induce cardiac hypertrophy, and mRNAs prepared from heart tissue were analyzed after 1, 6, and 42 days. The total concentration of HA was quantified, and HA and CD44 were studied histochemically. The expression of HAS1, HAS2, CD44, and FGF-2 was considerably up-regulated at days 1 and 6 and returned to basal levels after 42 days. FGFR-1 was up-regulated at day 1 but at basal levels once more at days 6 and 42. The concentration of HA significantly increased in aorta-ligated rats. Histochemical analysis showed increased expression of CD44 in hypertrophied myocardium mainly in and around the coronary arteries. These results agree well with other studies of tissue growth (malignancies and wound healing). The increase of HA, its synthases, and receptor in parallel with FGF-2 and its receptor illustrates their complicated interplay in the development of cardiac hypertrophy. The up-regulation of both HAS1 and HAS2 indicates the importance of HA production in the hypertrophic process and the possibility that HA is needed for two different purposes for the heart to be able to adapt to the increased afterload caused by aortic ligature. This research received financial support from the Swedish Heart Lung Foundation. The authors declare no conflicting financial interests.     

Hyaluronan suppresses prostate tumor cell proliferation through diminished expression of N-cadherin and aberrant growth factor receptor signaling

Hyaluronan (HA) production has been functionally implicated in prostate tumorigenesis and metastasis. We previously used prostate tumor cells overexpressing the HA synthesizing enzyme HAS3 or the clinically relevant hyaluronidase Hyal1 to show that excess HA production suppresses tumor growth, while HA turnover accelerates spontaneous metastasis from the prostate. Here, we examined pathways responsible for effects of HAS3 and Hyal1 on tumor cell phenotype. Detailed characterization of cell cycle progression revealed that expression of Hyal1 accelerated cell cycle re-entry following synchronization, whereas HAS3 alone delayed entry. Hyal1 expressing cells exhibited a significant reduction in their ability to sustain ERK phosphorylation upon stimulation by growth factors, and in their expression of the cyclin-dependent kinase inhibitor p21. In contrast, HAS3 expressing cells showed prolonged ERK phosphorylation and increased expression of both p21 and p27, in asynchronous and synchronized cultures. Changes in cell cycle regulatory proteins were accompanied by HA-induced suppression of N-cadherin, while E-cadherin expression and β-catenin expression and distribution remained unchanged. Our results are consistent with a model in which excess HA synthesis suppresses cell proliferation by promoting homotypic E-cadherin mediated cell–cell adhesion, consequently signaling to elevate cell cycle inhibitor expression and suppress G1- to S-phase transition.     

Natural antisense mRNAs to hyaluronan synthase 2 inhibit hyaluronan biosynthesis and cell proliferation

We report the identification of a natural antisense mRNA of hyaluronan synthase 2 that we have chosen to designate as HASNT (for HA synthase 2 antisense) in human and mouse. HASNT is transcribed from the opposite strand of the HAS2 gene locus and is represented by several independent expressed sequence tags in human. Portions of the mouse Hasnt gene were identified through an exon-trapping approach. Sequence conservation is extremely low between human and mouse HASNT, and it is not clear whether these mRNAs contain functional open reading frames. HASNT has an alternate splice site in both human and mouse. This splice site is located at an identical position within the gene in both species and results in mRNAs of two different lengths. In each species, the antisense portion of the HASNT gene is complementary to the first exon of HAS2, which represents the 5'-untranslated region. To study the biological activity of HASNT, two human expressed sequence tag clones, representing long and short HASNT splice variants, were cloned into a tetracycline-inducible vector and were stably transfected into human osteosarcoma U2-OS Tet-on cells. The long and short HASNT-expressing cells had a reduction in HAS2 mRNA levels up to 94 and 86%, respectively, whereas hyaluronan biosynthesis was inhibited by 40 and 37%, respectively. Cell proliferation was reduced throughout the time frame of the experiment. Exogenous high molecular mass hyaluronan failed to rescue the suppressed cell proliferation, whereas adenoviral-mediated overexpression of hyaluronan synthase 3, which stimulated endogenous hyaluronan biosynthesis, was able to rescue. Collectively, our data suggest that natural antisense mRNAs of HAS2 are able to regulate HAS2 mRNA levels and hyaluronan biosynthesis in a cell culture model system and may have an important and novel regulatory role in the control of HAS2, HA biosynthesis, and HA-dependent cell functions in vivo.     

The hyaluronan synthase catalyzes the synthesis and membrane translocation of hyaluronan

Hyaluronan (HA), an extracellular linear polysaccharide of alternating N-acetyl-glucosamine and glucuronic acid residues, is ubiquitously expressed in vertebrates, where it affects a broad spectrum of physiological processes, including cell adhesion, migration and differentiation. The HA polymer is synthesized on the cytosolic side of the cell membrane by the membrane-embedded hyaluronan synthase (HAS). However, the process by which the extremely hydrophilic HA polymer is translocated across the membrane is unknown to date. The bacterial HAS from Streptococcus equisimilis (Se) shares a similar transmembrane topology and significant sequence identity with human HASs and likely synthesizes HA by the same mechanism. We demonstrate that the Se-HAS is both necessary and sufficient to translocate HA in a reaction that is tightly coupled to HA elongation. The purified Se-HAS is reconstituted into proteoliposomes (PLs) where it synthesizes and translocates HA. In vitro synthesized, high-molecular-weight HA remains tightly associated with the intact PLs in sedimentation experiments. Most importantly, the newly formed HA is protected from enzymatic degradation by hyaluronidase unless the PLs are solubilized with detergent, thereby demonstrating that HA is translocated into the lumen of the vesicle. In addition, we show that HA synthesis and translocation are spatially coupled events, which allow HA synthesis even in the presence of a large excess of HA-degrading enzyme. The coupled synthesis and membrane translocation of a biopolymer represents a novel membrane translocation mechanism and is likely applicable to the synthesis of some of the most abundant biopolymers, including chitin and cellulose.     

Hyaluronic acid, HAS1, and HAS2 are significantly upregulated during muscle hypertrophy

Hyaluronic acid (HA) is a component of the extracellular matrix (ECM) in most vertebrate tissues and is thought to play a significant role during development, wound healing, and regeneration. In vitro studies have shown that HA enhances muscle progenitor cell recruitment and inhibits premature myotube fusion, implicating a role for this glycosaminoglycan in functional repair. However, the spatiotemporal distribution of HA during muscle growth and repair was unknown. We hypothesized that inducing hypertrophy via synergist ablation would increase the expression of HA and the HA synthases (HAS1-HAS3). We found that HA and HAS1-HAS3 were significantly upregulated within the plantaris muscle in response to Achilles tenectomy. HA concentration significantly increased 2.8-fold after 2 days but decreased towards levels comparable to age-matched controls by 14 days. Using immunohistochemistry, we found the colocalization of HAS1-HAS3 with macrophages, blood vessel epithelia, and fibroblasts varied in response to time and/or tenectomy. At the level of gene expression, only HAS1 and HAS2 significantly increased with respect to both time and tenectomy. The profiles of additional genes that influence ECM composition during muscle repair, tenascin-C, type I collagen, the HA-degrading hyaluronidases (Hyal) and matrix metalloproteinases (MMP) were also investigated. Hyal1 and Hyal2 were highly expressed in skeletal muscle but did not change after tenectomy; however, indicators of hypertrophy, MMP-2 and MMP-14, were significantly upregulated from 2 to 14 days. These results indicate that HA levels dynamically change in response to a hypertrophic stimulus and various cells may participate in this mechanism of skeletal muscle adaptation.     

Cleavage of hyaluronan is impaired in aged dermal wounds

Changes in extracellular matrix (ECM) are one of many components that contribute to impaired wound healing in aging. This study examined the effect of age on the glycosaminoglycan hyaluronan (HA) in normal and wounded dermis from young (4–6 month-old) and aged (22–24 month-old) mice. HA content and size were similar in the normal dermis of young and aged mice. Dermal explants labeled with [³H]-glucosamine showed decreased generation of smaller forms of HA in aged explants relative to young explants. Aged mice exhibited delayed wound repair compared with young mice with the greatest differential at 5 days. Expression of hyaluronan synthase (HAS) 2 and 3, and hyaluronidase (HYAL) 1–3 mRNA in wounds of young and aged mice was similar. There was a trend toward a decreased HYAL protein expression in aged wound dermis, which was accompanied by changes in detectable HYAL activity. Total HA content was similar in young and aged wound dermis. There was significantly less HA in the lower MW range (~ 250 kDa and smaller) in 5-day wound dermis, but not in 9-day wound dermis, from aged mice relative to young mice. We propose that decreased cleavage of HA is an additional component of impaired dermal wound healing in aging.