Abstract
Hyaluronan (HA) is synthesized by three HA synthases (HAS1, HAS2, and HAS3) and secreted in the extracellular matrix. In human skin, large amounts of HA are found in the dermis. HA is also synthesized by keratinocytes in the epidermis, although its epidermal functions are not clearly identified yet. To investigate HA functions, we studied the effects of HA depletion on human keratinocyte physiology within in vitro reconstructed human epidermis. Inhibition of HA synthesis with 4-methylumbelliferone (4MU) did not modify the expression profile of the epidermal differentiation markers involucrin, keratin 10, and filaggrin during tissue reconstruction. In contrast, when keratinocytes were incubated with 4MU, cell proliferation was decreased. In an attempt to rescue the proliferation function, HA samples of various mean molecular masses were added to keratinocyte cultures treated with 4MU. These samples were unable to rescue the initial proliferation rate. Furthermore, treatments with HA-specific hyaluronidase, although removing almost all HA from keratinocyte cultures, did not alter the differentiation or proliferation processes. The differences between 4MU and hyaluronidase effects did not result from differences in intracellular HA, sulfated glycosaminoglycan concentration, apoptosis, or levels of HA receptors, all of which remained unchanged. Similarly, knockdown of UDP-glucose 6-dehydrogenase (UGDH) using lentiviral shRNA effectively decreased HA production but did not affect proliferation rate. Overall, these data suggest that HA levels in the human epidermis are not directly correlated with keratinocyte proliferation and differentiation and that incubation of cells with 4MU cannot equate with HA removal.
Highlights
Golgi network; the molecule is assembled at the inner face of the plasma membrane, and the newly formed polymer is extruded into the extracellular matrix as it lengthens by addition of the dimer units
We have recently demonstrated that HAS1 is the main synthase involved in extracellular HA production by normal human keratinocytes, whereas HAS2 and HAS3 are instead implicated during the early steps of keratinocyte culture as well as in atopic dermatitis conditions [16]
Knockdown of CD44 expression in keratinocyte cultures using shRNA-bearing lentiviral particles did not influence cell proliferation (Fig. 2), ruling out an important role of CD44 in the control of human keratinocyte proliferation. These results suggest that the amount of HA in human keratinocyte monolayer cultures does not influence cell proliferation
Summary
Golgi network; the molecule is assembled at the inner face of the plasma membrane, and the newly formed polymer is extruded into the extracellular matrix as it lengthens by addition of the dimer units. In light of those conflicting data, the present study was designed to unravel the role of HA in proliferation and differentiation of human keratinocytes grown in a monolayer model and in an in vitro model that is as close as possible to native human epidermal growth, i.e. reconstructed human epidermis (RHE) cultured on a polycarbonate filter at an air-liquid interface.
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