Original research articleHyaluronan modulates growth factor induced mammary gland branching in a size dependent manner

- Remodeling mammary glands contain mixtures of HA polymer sizes (98% > 500 kDa and < 2% 7-114 kDa).
- 7-21 kDa HA fragments are highest during gestation and uniquely promote EGF induced branching morphogenesis in culture.
- High molecular weight HA suppresses the stimulatory effect of fragments.
- Fragment effects are mediated by CD44 and RHAMM.

Mammary gland morphogenesis begins during fetal development but expansion of the mammary tree occurs postnatally in response to hormones, growth factors and extracellular matrix. Hyaluronan (HA) is an extracellular matrix polysaccharide that has been shown to modulate growth factor-induced branching in culture. Neither the physiological relevance of HA to mammary gland morphogenesis nor the role that HA receptors play in these responses are currently well understood. We show that HA synthase (HAS2) is expressed in both ductal epithelia and stromal cells but HA primarily accumulates in the stroma. HA accumulation and expression of the HA receptors CD44 and RHAMM are highest during gestation when gland remodeling, lateral branch infilling and lobulo-alveoli formation is active. Molecular weight analyses show that approximately 98% of HA at all stages of morphogenesis is > 300 kDa. Low levels of 7-114 kDa HA fragments are also detected and in particular the accumulation of 7-21 kDa HA fragments are significantly higher during gestation than other morphogenetic stages (p < 0.05). Using these in vivo results as a guide, in culture analyses of mammary epithelial cell lines (EpH4 and NMuMG) were performed to determine the roles of high molecular weight, 7-21 kDa (10 kDa MWavg) and HA receptors in EGF-induced branching morphogenesis. Results of these assays show that while HA synthesis is required for branching and 10 kDa HA fragments strongly stimulate branching, the activity of HA decreases with increasing molecular weight and 500 kDa HA strongly inhibits this morphogenetic process. The response to 10 kDa HA requires RHAMM function and genetic deletion of RHAMM transiently blunts lateral branching in vivo. Collectively, these results reveal distinct roles for HA polymer size in modulating growth factor induced mammary gland branching and implicates these polymers in both the expansion and sculpting of the mammary tree during gestation.