Ng and motility (84, 85). The influence of hyaluronan on cell motility is
Ng and motility (84, 85). The influence of hyaluronan on cell motility is definitely complex. It has been shown to stimulate cell migration and in turn its removal MIG/CXCL9 Protein site inhibits migration (reviewed in Ref. 86). Nonetheless, hyaluronan can also inhibit cell motility, in particular when present in excessive amounts including through artificial overexpression induced by HAS transfection (87, 88). The physiological response from the cells to hyaluronan is determined by its molecular mass (24, 89). UTP induced mainly HAS2, that is known to synthesize high molecular mass hyaluronan (87, 90). No matter if hyaluronan Kallikrein-2, Human (HEK293, His) breakdown was activated by UTP was not investigated here. Nonetheless, we did not observe any enhance within the intracellular hyaluronan, which is usually linked with hyaluronan breakdown (31, 91). Moreover, the expression of hyaluronidases was not changed. Having said that, because the inhibition of cell motility occurs currently inside 15 to 30 min after the UTP exposure (84, 85), it unlikely relates to hyaluronan synthesis, which takes location later. Coordination and Convergence of Signaling in Keratinocyte Hyaluronan Responses Triggered by Injury and Environmental Stress–Interestingly, the UTP-induced signaling pathways regulating HAS2 expression partially overlap with those occurring just after UVB exposure, an insult known to result in nucleotide release in keratinocytes (9). While UVB induces a multitude of signaling events, in rat epidermal keratinocytes (REK) the induction of Has2 and Has3 appears to depend mostly on p38 and CaMKII (31). Even so, in REK cells UVB also activates the expression of other hyaluronan-related genes, for instance Has1, Hyal1, and Hyal2 (31), which didn’t respond to UTP. In addition, the UVB-induced activation of p38 and Has2 expression in rat keratinocytes is long-lasting (36 h), suggesting that for any far more sustained response other signaling pathways want to become activated. One of the UVB-activated cascades originates from HB-EGF/EGFR (92), shown to regulate Has2 and Has3 expresJOURNAL OF BIOLOGICAL CHEMISTRYExtracellular UTP Induces Hyaluronan Synthesission in REK cultures (32, 93). Even though the intracellular signaling mediators triggered by nucleotides and EGFR activation are partially distinct, they show convergence, suggesting that they could act synergistically (94). In conclusion, nucleotides UTP and UDP, but not UMP, released in to the extracellular space activate HAS2 mRNA expression in human keratinocytes. The expression with the other hyaluronan synthases or hyaluronidases will not be significantly influenced by UTP. The signaling pathway for UTP entails the purinergic P2Y2 receptor, and to a smaller extent the UDP receptors P2Y6 and P2Y14, and their downstream cascades recruiting PKC, plus the MAP kinases p38 and ERK, CaMKII, STAT3, and CREB (Fig. six). The impact of UTP on HAS2 expression is strong and, though transient, causes a considerable raise in hyaluronan accumulation in the pericellular matrix and culture medium. This fast induction of a hyaluronan coat can be a superb method to present an instant response to a potentially damaging signal, whereas making sure a stronger, additional sustained response is only activated if necessary by the situations.TABLE 1 Primer sequences for qRT-PCR in the human genesGene name ARP0 HAS1 HAS2 HAS3 HYAL1 HYAL2 P2Y2 Primer sequence (five to 3 ) Forword, AGATGCAGCAGATCCGCAT Reverse, GTGGTGATACCTAAAGCCTG Forward, CAAGATTCTTCAGTCTGGAC Reverse, TAAGAACGAGGAGAAAGCAG Forward, CAGAATCCAAACAGACAGTTC Reverse, TAAGGTGTTGTGTGTG.