Notes

1. The combined application of these in situ assays has allowed us to distinguish HSPGs that bind FGF-2 from those that bind FGF-7 (18), and to classify HSPGs as promoters and inhibitors of FGF-2 signaling complex formation (19). In human skin, an organ governed by signaling of different members of the FGF family, distinct differences are detected between keratinocyte surface HSPGs (likely members of the syndecan and glypican families of HSPGs) and epidermal basement membrane HSPGs (predominantly perlecan). Specifically, we find that keratinocyte surface HSPGs bind both FGF-2 and FGF-7, while epidermal basement membrane HSPGs bind FGF-2 but not FGF-7 (see Fig. 2B,C). Similarly, distinct differences between cell surface and basement membrane HSPGs are detected with regard to soluble FGF RTK binding. Epidermal surface HSPGs immobilize FR1c-AP, while epidermal basement membrane HSPGs failed to so (see Fig. 2E), despite the fact that HSPGs in this extracellular location bind ample amounts of FGF-2 (see Fig. 2C). These in situ techniques enable one to examine HSPG location and function in model systems where HSPGs have been found to be dynamically regulated and where FGFs play important roles, such as during development and in pathological conditions. The applicability of the assays to paraffin-embedded archival tissues opens the door to retrospective analysis of pathological specimens.

2. A few limitations of the in situ binding assays have to be emphasized. The assays do not allow the clear assignment of a certain HSPG species to FGF binding or signaling; they localize HS-binding activities and, when combined with immunohistochemical detection of HSPG core proteins, the HS may be shown to co-localize with a specific core protein. Also, it cannot be ruled out that endogenously produced HS-binding ligands may mask some of the binding sites on tissue HSPGs. In pilot experiments, we have rinsed tissue sections with 2 M NaCl before fixation to remove potential blocking ligands. No difference in binding patterns is detected, but this possibility cannot entirely be excluded. Despite these limitations, the ligand and soluble receptor binding assays provide important information on HSPG functions in vivo. These are tools to formulate hypotheses that can subsequently be vigorously tested with in vitro models.

Was this article helpful?

0 0
Healthy Chemistry For Optimal Health

Healthy Chemistry For Optimal Health

Thousands Have Used Chemicals To Improve Their Medical Condition. This Book Is one Of The Most Valuable Resources In The World When It Comes To Chemicals. Not All Chemicals Are Harmful For Your Body – Find Out Those That Helps To Maintain Your Health.

Get My Free Ebook


Post a comment