Raija Tammi

Parathyroid Hormone Rapidly Stimulates Hyaluronan Synthesis by Periosteal Osteoblasts in the Tibial Diaphysis of the Growing Rat

Short term treatment (3-24 h) with parathyroid hormone (PTH) stimulated the synthesis and accumulation of hyaluronan (HyA) in explant cultures of tibial diaphyses from young rats. PTH increased the overall HyA content of periosteum 5-fold, with the basal cambium layer exhibiting the greatest enhancement (∼8-fold). PTH increased the HyA content of cortical bone by 2-fold while not affecting the HyA content of bone marrow. PTH treatment greatly enhanced HyA staining throughout all layers of the periosteum, although its most dramatic effect occurred in the basal cambium layer. Here, unlike in the control tissue sections, nearly all cambium-lining osteoblasts stained intensely positive for HyA. PTH treatment enhanced the HyA staining of osteocytes in cortical bone tissue sections to the extent that the lacunocanalicular system became visualized. Three significant findings were revealed in this study. First, mature periosteal osteoblasts, under natural conditions, do not contain much HyA in their surrounding extracellular matrix but dramatically enhance their matrix HyA content when treated with PTH. Second, pre-osteocytes and osteocytes contain more HyA in their natural matrix than mature lining osteoblasts, and they appear to have functional PTH receptors because they responded to PTH treatment with an enhancement of HyA content. Finally, it was observed that the lining cells along the endosteal surface of the diaphysis did not stain strongly positive for HyA either naturally or when exposed to PTH treatment. This indicates that periosteal and endosteal osteoblastic cell populations exhibit metabolic differences in their extracellular matrix responses to PTH.

Chapter 19 – Hyaluronan in the Epidermis and Other Epithelial Tissues

Chemical assays indicate that skin is the largest single source of hyaluronan in the bodies of humans and other mammals. The dermal connective tissue constitutes the bulk of the skin, while epidermis—the epithelial compartment—is thinner than 50 μm in most locations of the human body surface. Hyaluronan in the human epidermis is mostly extracellular, localized between the basal and spinous cells, that is, the live cell layers. In contrast, when keratinocytes enter the terminal differentiation program and go to the granular cell stage and later develop the cornified cells, no hyaluronan signal remains detectable for the specific histological probe that recognizes hyaluronan. Interestingly, hyaluronan signal is also missing from the basal side of the basal cells in normal human epidermis. The epidermal localization of the hyaluronan receptor CD44 completely overlaps with that of hyaluronan; both reside in the pouches between desmosomes connecting adjacent keratinocytes. The hyaluronan deposits in the epidermis are excluded from structures with tight adherence to adjacent cells or matrix, suggesting that its synthesis is specifically directed to places on the plasma membrane where open extracellular space is needed. The currently available data on hyaluronan metabolism in the epidermis is derived from metabolic labeling of pig and human skin explants in organ culture and organotypic, differentiating rat keratinocyte cultures. Analysis of 3H-glucosamine incorporation revealed that hyaluronan was the major glycosaminoglycan synthesized in human epidermis.

Hyaluronan in Human Tumors

Publisher Summary nHyaluronan has turned out to be an active regulator of cell behavior rather than solely an inert extracellular matrix component. The high concentration of hyaluronan in many embryonic tissues correlates with their rates of cell migration and proliferation. Recent reports suggest that hyaluronan is not only a prognostic indicator, but also an active participant in the disease and a novel target of therapy. There is a large body of evidence from experimental animals and in vitro models suggesting that the production of hyaluronan by tumor cells is important for their malignant behavior. The assays on tumor biopsies do not give information about the dynamic processes that control the synthesis and catabolism of hyaluronan. Analyses of clinical patient materials show that alterations in the hyaluronan content, whether on the actual malignant cells or their surrounding stroma, are tightly associated with patient prognosis. Tumor progression is highly likely when hyaluronan is abundant on the surface or within tumor cells of gastric or colon carcinoma. There are number of ways hyaluronan can be involved in the regulation of cancer growth and spreading, as suggested by experiments on animals, and studies in vitro. However, understanding the relative importance of the various aspects of hyaluronan functions and metabolism in human cancers in vivo is still lacking and warrants more research on clinical materials.Publisher Summary Hyaluronan has turned out to be an active regulator of cell behavior rather than solely an inert extracellular matrix component. The high concentration of hyaluronan in many embryonic tissues correlates with their rates of cell migration and proliferation. Recent reports suggest that hyaluronan is not only a prognostic indicator, but also an active participant in the disease and a novel target of therapy. There is a large body of evidence from experimental animals and in vitro models suggesting that the production of hyaluronan by tumor cells is important for their malignant behavior. The assays on tumor biopsies do not give information about the dynamic processes that control the synthesis and catabolism of hyaluronan. Analyses of clinical patient materials show that alterations in the hyaluronan content, whether on the actual malignant cells or their surrounding stroma, are tightly associated with patient prognosis. Tumor progression is highly likely when hyaluronan is abundant on the surface or within tumor cells of gastric or colon carcinoma. There are number of ways hyaluronan can be involved in the regulation of cancer growth and spreading, as suggested by experiments on animals, and studies in vitro. However, understanding the relative importance of the various aspects of hyaluronan functions and metabolism in human cancers in vivo is still lacking and warrants more research on clinical materials.

Hyaluronan in Human Tumors: Importance of Stromal and Cancer Cell-Associated Hyaluronan

Publisher Summary nHyaluronan has turned out to be an active regulator of cell behavior rather than solely an inert extracellular matrix component. The high concentration of hyaluronan in many embryonic tissues correlates with their rates of cell migration and proliferation. Recent reports suggest that hyaluronan is not only a prognostic indicator, but also an active participant in the disease and a novel target of therapy. There is a large body of evidence from experimental animals and in vitro models suggesting that the production of hyaluronan by tumor cells is important for their malignant behavior. The assays on tumor biopsies do not give information about the dynamic processes that control the synthesis and catabolism of hyaluronan. Analyses of clinical patient materials show that alterations in the hyaluronan content, whether on the actual malignant cells or their surrounding stroma, are tightly associated with patient prognosis. Tumor progression is highly likely when hyaluronan is abundant on the surface or within tumor cells of gastric or colon carcinoma. There are number of ways hyaluronan can be involved in the regulation of cancer growth and spreading, as suggested by experiments on animals, and studies in vitro. However, understanding the relative importance of the various aspects of hyaluronan functions and metabolism in human cancers in vivo is still lacking and warrants more research on clinical materials.Publisher Summary Hyaluronan has turned out to be an active regulator of cell behavior rather than solely an inert extracellular matrix component. The high concentration of hyaluronan in many embryonic tissues correlates with their rates of cell migration and proliferation. Recent reports suggest that hyaluronan is not only a prognostic indicator, but also an active participant in the disease and a novel target of therapy. There is a large body of evidence from experimental animals and in vitro models suggesting that the production of hyaluronan by tumor cells is important for their malignant behavior. The assays on tumor biopsies do not give information about the dynamic processes that control the synthesis and catabolism of hyaluronan. Analyses of clinical patient materials show that alterations in the hyaluronan content, whether on the actual malignant cells or their surrounding stroma, are tightly associated with patient prognosis. Tumor progression is highly likely when hyaluronan is abundant on the surface or within tumor cells of gastric or colon carcinoma. There are number of ways hyaluronan can be involved in the regulation of cancer growth and spreading, as suggested by experiments on animals, and studies in vitro. However, understanding the relative importance of the various aspects of hyaluronan functions and metabolism in human cancers in vivo is still lacking and warrants more research on clinical materials.

HYALURONAN SYNTHASE 2 (HAS2) REGULATES MIGRATION OF EPIDERMAL KERATINOCYTES

ABSTRACT Hyaluronan (HA) is a linear polysaccharide abundant in the extracellular space between epidermal keratinocytes. It is synthesized at the inner face of the plasma membrane by hyaluronan synthases (Has). We probed the importance of hyaluronan in keratinocytes by establishing cell lines carrying exogenous hyaluronan synthase 2 (Has2) gene(s) in sense and antisense orientations in order to increase and decrease their hyaluronan synthesis, respectively. The cell lines with the sense Has2 cDNA showed increased HA synthesis, while most cell lines with Has2 antisense cDNA contained less HA. Has2 antisense cells differed from control cell lines; they spread at a slower rate and retained a rounded morphology for a longer time. Further, during the first 24 hours after plating, proliferation was delayed in the antisense cell lines. In an in vitro wounding assay the antisense cells showed a significantly decreased migration rate as compared to controls. Cell lines with the Has2 sense cDNA were similar to the control cell lines in spreading and proliferation rates. However, they migrated faster than control cell lines.

CHAPTER 14 – Hyaluronan in Human Tumors: Importance of Stromal and Cancer Cell-Associated Hyaluronan

Publisher Summary nHyaluronan has turned out to be an active regulator of cell behavior rather than solely an inert extracellular matrix component. The high concentration of hyaluronan in many embryonic tissues correlates with their rates of cell migration and proliferation. Recent reports suggest that hyaluronan is not only a prognostic indicator, but also an active participant in the disease and a novel target of therapy. There is a large body of evidence from experimental animals and in vitro models suggesting that the production of hyaluronan by tumor cells is important for their malignant behavior. The assays on tumor biopsies do not give information about the dynamic processes that control the synthesis and catabolism of hyaluronan. Analyses of clinical patient materials show that alterations in the hyaluronan content, whether on the actual malignant cells or their surrounding stroma, are tightly associated with patient prognosis. Tumor progression is highly likely when hyaluronan is abundant on the surface or within tumor cells of gastric or colon carcinoma. There are number of ways hyaluronan can be involved in the regulation of cancer growth and spreading, as suggested by experiments on animals, and studies in vitro. However, understanding the relative importance of the various aspects of hyaluronan functions and metabolism in human cancers in vivo is still lacking and warrants more research on clinical materials.Publisher Summary Hyaluronan has turned out to be an active regulator of cell behavior rather than solely an inert extracellular matrix component. The high concentration of hyaluronan in many embryonic tissues correlates with their rates of cell migration and proliferation. Recent reports suggest that hyaluronan is not only a prognostic indicator, but also an active participant in the disease and a novel target of therapy. There is a large body of evidence from experimental animals and in vitro models suggesting that the production of hyaluronan by tumor cells is important for their malignant behavior. The assays on tumor biopsies do not give information about the dynamic processes that control the synthesis and catabolism of hyaluronan. Analyses of clinical patient materials show that alterations in the hyaluronan content, whether on the actual malignant cells or their surrounding stroma, are tightly associated with patient prognosis. Tumor progression is highly likely when hyaluronan is abundant on the surface or within tumor cells of gastric or colon carcinoma. There are number of ways hyaluronan can be involved in the regulation of cancer growth and spreading, as suggested by experiments on animals, and studies in vitro. However, understanding the relative importance of the various aspects of hyaluronan functions and metabolism in human cancers in vivo is still lacking and warrants more research on clinical materials.

Hyaluronan stimulates keratinocyte migration and activates the transcription factor AP-1 in keratinocytes through the JNK pathway

Hyaluronan (HA) has been considered a passive extracellular matrix (ECM) polysaccharide, but recent studies have shown its importance in controlling many cell functions including motility, proliferation and adhesion, which imply signaling from ECM to cytosol. Hyaluronan is a major ECM component in stratified epithelia such as skin epidermis. We found that hyaluronan added to the growth medium of newly plated human skin keratinocytes increased cell migration in an in vitro wound-healing assay. Hyaluronan also increased the transcription factor AP-1, as determined by gel shift assays. The kinase signals that apperently led to the increased AP-1 level were associated with the activation of c-Jun, mainly via the JNK pathway as early as 10 min after the addition of hyaluronan, and with the minimum concentration of 10 ng/ml. ERK1 was also slightly activated, while p38 MAPkinase was not affected.

EGF REGULATES HAS2 EXPRESSION, CONTROLS EPIDERMAL THICKNESS AND STIMULATES KERATINOCYTE MIGRATION

High concentrations of hyaluronan reside in the small space between the vital kertinocyte layers of human and animal epidermis and influence keratinocyte interactions, including growth, mobility and differentiation. We have previously found that the content of epidermal hyaluronan in human skin organ cultures is decreased and increased by cortisol and retinoic acid, and associated with enhanced and retarded terminal differentiation, respectively. To further substantiate this idea, we incubated epidermal keratinocytes with epidermal growth factor (EGF), and found a marked increase in hyaluronan synthesis which correlated with faster migration in an in vitro wounding assay of keratinocyte monolayers. EGF increased hyaluronan also in stratified, differentiated organotypic cultures, and increased the height of vital epidermis and reduced the thickness of the cornified layers, findings in line with an inhibition of terminal differentiation of keratinocytes. The stimulation of hyaluronan synthesis by EGF was due to upregulation of hyaluronan synthase 2 (HAS2) but not HAS1 or HAS3. A part of the EGF influence on the structure of epidermis, and on skin wound healing, is thus mediated through its control of HAS2 expression.

INTRACELLULAR HYALURONAN IN EPIDERMAL KERATINOCYTES

ABSTRACT Rat epidermal keratinocyte monolayer cultures (REKs) actively synthesize hyaluronan (HA) most of which is retained on the cell surface or released into culture medium. However, a small proportion of HA also resides in an intracellular compartment (IC-HA). We characterized IC-HA localization and processing in REKs using specific staining with HA-binding probe, and its size by gel filtration of metabolically labeled HA. About 3% of REKs exhibited abundant IC-HA, while half of the cells lacked any microscopically demonstrable IC-HA. IC-HA was localized in 200–600 nm cytoplasmic vesicles. Dual staining of IC-HA with markers for lysosomes showed no colocalization. When REKs were treated with HA10 (decasaccharide), all IC-HA disappeared, while HA6, HA4, and sulfated glycosaminoglycans had no effect. All IC-HA was cleared 10 min after addition of HA10, and a 50%-reduction was reached in 5 min. An anti-CD44 mab, that increases HA on the cell surface, also increased IC-HA. Inhibiton of endocytosis via coated pits and caveolae did not reduce the amount of IC-HA. Perturbation of lysosomal activity caused accumulation of IC-HA. Most of the IC-HA had low MW ( 2000 kDa). The data demonstrate a rapid uptake and lysosomal degradation of IC-HA in REKs via a receptor dependent route separate from coated pits and caveolae, that involves a receptor with HA10 specificity, identified as CD44 or functionally dependent on CD44. The IC-HA consists of small HA fragments which may have a specific role in cellular homeostasis.

HYALURONAN METABOLISM AND DISTRIBUTION IN STRATIFIED, DIFFERENTIATED CULTURES OF EPIDERMAL KERATINOCYTES

ABSTRACT Hyaluronan is a major intercellular matrix molecule in the vital cell layers of skin epidermis. An organotypic culture model was developed to study epidermal hyaluronan metabolism by using a rat epidermal keratinocyte line (REK) which exhibits histodifferentiation similar to that of the native epidermis when cultured at an air-liquid interface. Two different support matrices were used: reconstituted collagen fibrils with and without a covering basal lamina previously deposited by canine kidney cells. REKs formed a stratified squamous, keratinized epithelium on both support matrices. Hyaluronan and its receptor, CD44, colocalized in the basal and spinous layers similar to their distribution in the native epidermis. Most (∼75%) of the newly synthesized hyaluronan was retained in the epithelium when a basal lamina was present while most (∼80%) diffused out of the epithelium in its absence. While REKs on the two matrices synthesized hyaluronan at essentially the same rate, catabolism of the macromolecules was much higher in the epithelium on the basal lamina with a half-life of approximately one day, similar to its half-life in native human epidermis. The formation of a true epidermal compartment bounded by the cornified layer on the surface and the basal lamina subjacent to the basal cells provides a good model within which to study the regulation of synthesis and catabolism of epidermal hyaluronan, isolated from the influence of dermal cells, or supporting cells (feeder cells) included in a collagen matrix.