Cytokines, e.g., interleukins interferon γ     Collagens,   fibronectin,   laminin.

       Lipoprotein lipaseThe majority of growth factors and morphogens implicated in the regulation of mammary gland development bind to HS (Table 1) and it appears that each of these proteins recognises a different structure in HS. In addition, key regulatory binding proteins such as IGFBP3 and IGFBP5 also bind to HS. The three main components of the extracellular matrix and the basement membrane, collagens, fibronectin and laminin, also interact with specific S-domains in HS, though it would appear that the collagens collectively recognise saccharide sequences of similar, if not identical structure. Finally, lipoprotein lipase, a key enzyme in lipid metabolism, which is an important feature of lactation is anchored to its target cells through HS.

2.  REGULATORY ACTIVITIES OF HS

      HS critically controls two key features of extracellular regulatory proteins, their localisation and sequestration and their generation of intracellular signals.The sequestration of a growth factor on extracellular HS was first proposed 12 years ago. This can prevent the diffusion of the growth factor within a tissue compartment or between tissue compartments, as well as allowing a local store of the growth factor to act on a restricted number of cells. In the mammary gland there is direct evidence for HS performing this function with regard to FGF-2 and FGF-3, and indirect evidence for many other HS-binding proteins. A regulatory role for HS is supported by the observation of changes in the structure of the HS chains which accompany the growth and development of many epithelia, including the mammary gland. Moreover, the association between the growth state of the cells (quiescent duct versus growing terminal end bud) and the number of HS receptors for FGF-2 associated with these structures in vivo suggests that HS dynamically regulates the activity of FGF-2 in the developing mammary gland. Other results support a similar role for HS in the regulation of the activity of HGF/SF and Wnt family members and it is likely from studies in other systems that HS has an analogous function towards all the growth factors and cytokines in Table 1.
      Many of the HS-binding proteins possess a dual receptor system, which consists of a conventional signalling receptor, e.g., receptor tyrosine kinase or integrin and an HS receptor. Upon ligand binding the conventional signalling receptor activates specific intracellular signalling pathways. The dual receptor systems provide a means to increase the combinatorial outcome of cellular signals generated by a single effector.

       FGF-2 is the classic growth factor with a dual receptor system. It elicits a cellular response through its FGFR receptor tyrosine kinase. In the presence of the activating HS receptor, a specific temporal pattern of activation of signalling pathways is observed. For example, following the addition of FGF-2 to Go synchronised rat mammary (Rama) 27 fibroblasts, the phosphorylation of mitogen-activated protein kinase (MAPK) peaks after 10 min, and then declines to half maximal levels, which are maintained well into S-phase, 18 h later. The early peak of MAPK phosphorylation is probably related to the activation of the c-fos promoter. In the same cells treated with chlorate, which prevents the biosynthesis of the HS receptor, the signalling pathways are activated in a transient manner, which is sufficient for the stimulation of c-fos transcription, but not for entry into the cell cycle.

        HGF/SF, a product of the mammary stroma, has been thought to target exclusively the epithelium. Indeed HGF/SF does not stimulate the growth of Rama 27 fibroblasts and these cells possess very few HS receptors for HGF/SF. However, Rama 27 fibroblasts do produce c-met, the tyrosine kinase receptor for HGF/SF. In the absence of its HS receptor, HGF/SF stimulates the phosphorylation of MAPK in Rama 27 fibroblasts biphasically, a response that is neither sustained nor sufficient for cell proliferation.

       There is also evidence for a contribution of the proteoglycan core protein to dual receptor systems. Human mammary (Huma) myoepithelial-like 109 cells possess both the HS18 and the c-met receptors for HGF. When these cells are grown on a plastic substratum, they express the proteoglycans perlecan and syndecan-2 on their cell surfaces and, surprisingly, HGF/SF has no effect on the proliferation of these cells. However, under these conditions HGF/SF does cause an early and transient stimulation of the phosphorylation of MAPK. In contrast, HGF/SF induces a sustained activation of MAPK and stimulates the proliferation of Huma 109 cells grown on a fibronectin or collagen substratum. Compared to cells grown on a plastic substratum, Huma   109   myoepithelial-like   cells   grown   on   fibronectin   or   collagen substrata show a marked reduction in the expression of cell surface perlecan, but not syndecan-2.There are, as yet, no studies in transgenic mice in which a perturbation to HS biosynthesis in the mammary gland has been introduced. A number of studies have examined the effects of chlorate, an inhibitor of the sulphation of HS on the differentiation of mammary epithelial cells in collagen gels. The results show that both the basal morphogenic potential of the cells and the ability of FGFs and HGF/SF to stimulate ductal morphogenesis in this system are dependent on HS.