Identification of a distal RXFP1 gene enhancer with differential activity in fibrotic lung fibroblasts involving AP-1.

Relaxin/insulin-like family peptide receptor 1 (RXFP1) mediates relaxin's antifibrotic effects and has reduced expression in the lung and skin of patients with fibrotic interstitial lung disease (fILD) including idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). This may explain the failure of relaxin-based anti-fibrotic treatments in SSc, but the regulatory mechanisms controlling RXFP1 expression remain largely unknown. This study aimed to identify regulatory elements of RXFP1 that may function differentially in fibrotic fibroblasts. We identified and evaluated a distal regulatory region of RXFP1 in lung fibroblasts using a luciferase reporter system. Using serial deletions, an enhancer upregulating pGL3-promoter activity was localized to the distal region between -584 to -242bp from the distal transcription start site (TSS). This enhancer exhibited reduced activity in IPF and SSc lung fibroblasts. Bioinformatic analysis identified two clusters of activator protein 1 (AP-1) transcription factor binding sites within the enhancer. Site-directed mutagenesis of the binding sites confirmed that only one cluster reduced activity (-358 to -353 relative to distal TSS). Co-expression of FOS in lung fibroblasts further increased enhancer activity. In vitro complex formation with a labeled probe spanning the functional AP-1 site using nuclear proteins isolated from lung fibroblasts confirmed a specific DNA/protein complex formation. Application of antibodies against JUN and FOS resulted in the complex alteration, while antibodies to JUNB and FOSL1 did not. Analysis of AP-1 binding in 5 pairs of control and IPF lung fibroblasts detected positive binding more frequently in control fibroblasts. Expression of JUN and FOS was reduced and correlated positively with RXFP1 expression in IPF lungs. In conclusion, we identified a distal enhancer of RXFP1 with differential activity in fibrotic lung fibroblasts involving AP-1 transcription factors. Our study provides insight into RXFP1 downregulation in fILD and may support efforts to reevaluate relaxin-based therapeutics alongside upregulation of RXFP1 transcription.

Native freshwater species get out of the way: Prussian carp (Carassius gibelio) impacts both fish and benthic invertebrate communities in North America.

Prussian carp (Carassius gibelio) are one of the most noxious non-native species in Eurasia. Recently, Prussian carp, a non-native freshwater fish species, were genetically confirmed in Alberta, Canada and have been rapidly expanding their range in North America since establishment. Given their rapid range expansion, there is an increasing need to determine how Prussian carp may impact native species. We assessed the severity of the Prussian carp invasion by (i) determining their impact on fish communities, (ii) assessing their impact on benthic invertebrate communities, (iii) evaluating if Prussian carp alter abiotic conditions, and (iv) identifying where we find higher abundances of Prussian carp. When Prussian carp were established, we found significant changes to the fish community. Correspondingly, the degree of impact to benthic invertebrate communities was related to the stage of invasion (none, early or recent), where changes in fish communities were significantly concordant with changes in benthic invertebrate communities. Finally, we found that higher abundances of Prussian carp were significantly associated with lower abundances of a majority of native fish species. Altogether, using three lines of evidence, we determine that Prussian carp can have wide-ranging impacts on freshwater ecosystems in North America, pressing the need for management intervention.