MicroRNA-7 inhibition rescues age-associated loss of epidermal growth factor receptor and hyaluronan-dependent differentiation in fibroblasts.

Age-related defects in fibroblast differentiation were previously shown to be associated with impaired hyaluronan synthase 2 (HAS2) and epidermal growth factor receptor (EGFR) function, with both required for normal fibroblast functionality. In fibroblasts, transforming growth factor-beta 1 (TGF-β1)-dependent phenotypic activation uses two distinct but co-operating pathways that involve TGF-β receptor (TGF-βR)/Smad2 activation and HA-mediated CD44-EGFR co-localization and signalling through extracellular signal-regulated kinase 1/2 (ERK1/2). The HA-mediated CD44-EGFR pathway was found to be compromised with in vitro aging, through loss of EGFR expression and a reduced movement of CD44 throughout the cellular membrane. Here, we also investigate the involvement of microRNAs (miRNAs) in age-related loss of differentiation, through investigation of miRNA-7 (miR-7) regulation of the HA-mediated EGFR-signalling pathway. The transcription of miR-7 was found to be upregulated in aged cells. In young cells, age-related loss of differentiation could be mimicked through transfection of pre-miR-7, and in aged cells, could be reversed through transfection of locked nucleic acids (LNA) targeting miR-7. Additionally, miR-7 was found to be involved in the regulation of CD44 membrane motility, which was downregulated in instances of miR-7 upregulation, and partially restorable through either miR-7 inhibition or HAS2 overexpression. The altered dynamics of CD44 in the cell membrane demonstrated a further action of miR-7 in regulating the HA-dependent CD44/EGFR pathway. We explain this novel mechanism of age-associated functional consequence due to miR-7 upregulation and demonstrate that it is reversible; highlighting miR-7 as a potential target for restoring the healing capabilities in chronic wounds in the elderly.

Midgley AC ，Bowen T ，Phillips AO ，Steadman R ... -  《-》

Aging fibroblasts resist phenotypic maturation because of impaired hyaluronan-dependent CD44/epidermal growth factor receptor signaling.

Simpson RM ，Wells A ，Thomas D ，Stephens P ，Steadman R ，Phillips A ... -  《-》

Transforming growth factor-β1 (TGF-β1)-stimulated fibroblast to myofibroblast differentiation is mediated by hyaluronan (HA)-facilitated epidermal growth factor receptor (EGFR) and CD44 co-localization in lipid rafts.

Fibroblast to myofibroblast differentiation drives effective wound healing and is largely regulated by the cytokine transforming growth factor-β1 (TGF-β1). Myofibroblasts express α-smooth muscle actin and are present in granulation tissue, where they are responsible for wound contraction. Our previous studies show that fibroblast differentiation in response to TGF-β1 is dependent on and mediated by the linear polysaccharide hyaluronan (HA). Both the HA receptor, CD44, and the epidermal growth factor receptor (EGFR) are involved in this differentiation response. The aim of this study was to understand the mechanisms linking HA-, CD44-, and EGFR-regulated TGF-β1-dependent differentiation. CD44 and EGFR co-localization within membrane-bound lipid rafts was necessary for differentiation, and this triggered downstream mitogen-activated protein kinase (MAPK/ERK) and Ca(2+)/calmodulin kinase II (CaMKII) activation. We also found that ERK phosphorylation was upstream of CaMKII phosphorylation, that ERK activation was necessary for CaMKII signaling, and that both kinases were essential for differentiation. In addition, HA synthase-2 (HAS2) siRNA attenuated both ERK and CaMKII signaling and sequestration of CD44 into lipid rafts, preventing differentiation. In summary, the data suggest that HAS2-dependent production of HA facilitates TGF-β1-dependent fibroblast differentiation through promoting CD44 interaction with EGFR held within membrane-bound lipid rafts. This induces MAPK/ERK, followed by CaMKII activation, leading to differentiation. This pathway is synergistic with the classical TGF-β1-dependent SMAD-signaling pathway and may provide a novel opportunity for intervention in wound healing.

Midgley AC ，Rogers M ，Hallett MB ，Clayton A ，Bowen T ，Phillips AO ，Steadman R ... -  《-》

Hyaluronan facilitates transforming growth factor-β1-dependent proliferation via CD44 and epidermal growth factor receptor interaction.

Fibroblast proliferation is an early feature of progressive tissue fibrosis and is largely regulated by the cytokine transforming growth factor-β1 (TGF-β1). In the oral mucosa, fibroblasts have a unique phenotype and demonstrate healing with no fibrosis/scarring. Our previous studies show that whereas dermal fibroblasts proliferate in response to TGF-β1, oral fibroblasts have an antiproliferative response to this cytokine. Hyaluronan (HA) was directly linked to this TGF-β1-dependent response. The aim of this study was to understand the underlying mechanism through which HA regulates TGF-β-dependent responses. Using patient-matched oral and dermal fibroblasts, we show that TGF-β1-dependent proliferation is mediated through the HA receptor CD44, whereas the TGF-β1-mediated antiproliferative response is CD44-independent. Furthermore, overexpression of HAS2 (HA synthase-2) in oral cells modifies their response, and they subsequently demonstrate a proliferative, CD44-dependent response to TGF-β1. We also show that epidermal growth factor (EGF) and its receptor (EGFR) are essential for TGF-β1/HA/CD44-dependent proliferation. Increased HA levels promote EGFR and CD44 coupling, potentiating signal transduction through the MAPK/ERK pathway. Thus, in a HA-rich environment, late ERK1/2 activation results from EGFR/CD44 coupling and leads to a proliferative response to TGF-β1. In comparison, in a non-HA-rich environment, only early ERK1/2 activation occurs, and this is associated with an antiproliferative response to TGF-β1. In summary, HA facilitates TGF-β1-dependent fibroblast proliferation through promoting interaction between CD44 and EGFR, which then promotes specific MAPK/ERK activation, inducing cellular proliferation.

Meran S ，Luo DD ，Simpson R ，Martin J ，Wells A ，Steadman R ，Phillips AO ... -  《-》

17β-estradiol ameliorates age-associated loss of fibroblast function by attenuating IFN-γ/STAT1-dependent miR-7 upregulation.

Age-related defects in fibroblast differentiation and functionality were previously shown to be associated with impaired hyaluronan (HA) synthase 2 (HAS2) and epidermal growth factor receptor (EGFR) function, as a result of upregulated microRNA-7 (miR-7) expression. In aging fibroblasts, inhibiting miR-7 prevented the dysregulation of the HA-mediated CD44/EGFR signaling pathway. Here, we investigated transcriptional upregulation of miR-7 and implicated the age-associated over-activation of JAK/STAT1 as a primary candidate. STAT1 binding sites were identified on the putative miR-7 promoter and stimulation of fibroblasts with the inflammatory cytokine, interferon-γ (IFN-γ), significantly increased miR-7 transcriptional activity and resulted in upregulated miR-7 and loss of EGFR. Additionally, we demonstrated a role for the anti-inflammatory steroid, 17β-estradiol (E2), in the attenuation of miR-7 expression. E2 stimulation promoted estrogen receptor (ER) interactions with the miR-7 putative promoter and suppressed miR-7 expression. E2 also attenuated STAT1 expression and activity. Furthermore, treatments with E2 restored fibroblast functionality, including proliferation, migration and differentiation, key events in effective wound healing. In light of our findings, we propose that the regulation of miR-7 by pro- and anti-inflammatory mediators plays a wider role than previously thought. The modulation of fibroblast functions and ultimately wound healing by miR-7 activators or inhibitors could provide realistic targets for the restoration of chronic wound healing capabilities in the elderly.

Midgley AC ，Morris G ，Phillips AO ，Steadman R ... -  《-》