Hyaluronan (HA) is a large but simple glycosaminoglycan composed of repeating D-glucuronic acid, beta1-3 linked to N-acetyl-D-glucosamine beta1-4, found in body fluids and tissues, in both intra- and extracellular compartments. Despite its structural simplicity, HA has diverse functions in skeletal biology. In development, HA-rich matrices facilitate migration and condensation of mesenchymal cells, and HA participates in joint cavity formation and longitudinal bone growth. In adult cartilage, HA binding to aggrecan immobilises aggrecan, retaining it at the high concentrations required for compressive resilience. HA also appears to regulate bone remodelling by controlling osteoclast, osteoblast and osteocyte behaviour. The functions of HA depend on its intrinsic properties, which in turn rely on the degree of polymerisation by HA synthases, depolymerisation by hyaluronidases, and interactions with HA-binding proteins. HA synthesis and degradation are closely regulated in skeletal tissues and aberrant synthetic or degradative activity causes disease. The role and regulation of HA synthesis and degradation in cartilage, bone and skeletal development is discussed.

It is well established that local modification of extracellular matrix (ECM) hyaluronan composition is vital in the regulation of cell behavior. Indeed, the formation of articulating chick joint cavities, which requires mechanical stimuli derived from skeletal movement, is dependent upon the accumulation of an ECM rich in hyaluronan (HA). However, the mechanisms responsible for such precise mechano-dependent regulation of cell behavior and the formation of a HA-rich ECM remain undefined. Here we show that extracellular-regulated kinase 1/2 (ERK1/2) is selectively activated in cells at sites of cavity formation and activity diminished by in ovo immobilization that induces cartilaginous fusion across presumptive joint interzones. In vitro analyses offer mechanistic support for the role of mechanical stimuli in promoting a MEK-dependent activation of ERK1/2. In addition, our direct regulation of ERK1/2 phosphorylation status via modulation of its up-stream "classical cascade" activator either pharmacologically or by transfection with dominant negative or constitutively active Mek confirms the essential role for ERK1/2 activation in the elaboration of HA-rich pericellular matrices. Together, our findings demonstrate that the MEK-ERK pathway, regulated by mechanical stimuli, controls HA-rich matrix assembly. The precision of ERK1/2 activation selectively distinguishing cells at the joint line suggests that it directly contributes to the loss of tissue cohesion essential for generating HA-rich cavities between joint elements during their development.

Mammary epithelial cells that produce milk are unique in containing both of the Cu pumps, ATP7A and B, central to mammalian copper homeostasis but otherwise found individually. We have begun studies to clarify their roles in this gland using confocal microscopy. ATP7B was expressed in the luminal epithelial cells of human breast tissue, with a perinuclear location that shifted to a diffuse location with lactation. In the human mammary epithelial cell culture model (PMC42), ATP7B was also in the perinuclear region and redistributed to endosomes adjacent to, but not coincident with, the apical plasma membrane after treatment of the cultures with a lactational hormone regimen (3 days estrogen and progesterone then 3 days insulin, dexamethasone, prolactin). ATP7B was in a different subset of vesicles from those containing milk proteins and did not overlap with endosomes containing ATP7A. A physiological concentration of extracellular Cu was required for the redistribution effect, suggesting it was caused indirectly by increased intracellular Cu concentrations. Overexpression of ATP7B in the PMC42 cultures, grown as polarized monolayers, resulted in an enhanced efflux of Cu from the apical cell surface (determined with 64Cu). Our findings are consistent with the concept that ATP7B is important for the secretion of copper from the mammary gland into the milk. Supported by PHS Grant No. RO1 HD 46949.