qPCR primers.

<p>qPCR primers.</p

Dose response for 2MbisP, CAGE-3 and atRA on utricle area and epidermal thickness.

<p>Utricle area and epidermal thickness were measured in H&E stained tissue sections after the application of 7 doses of vehicle or varying doses of 2MbisP (A, B), CAGE-3 (C, D), or atRA (E, F). Significant differences from the vehicle group at each respective dose are indicated by an asterisk, *<i>P</i>≤0.05 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188887#pone.0188887.s002" target="_blank">S2 Table</a>).</p

EGFR ligand mRNA after 7 topical doses of 2MbisP, CAGE-3 or atRA.

<p>EGFR ligand mRNAs were analyzed by RT-PCR in skin taken 4 h after receiving the final dose of vehicle, 2MbisP (690 nmol/kg), CAGE-3 (0.25 nmol/kg), or atRA (224 nmol/kg) and the data are expressed relative to the respective vehicle-treated group (treatment/vehicle).</p

Structure of 1α,25(OH)₂D₃ compared to the 2-methylene-19-nor analogs, 2MbisP and CAGE-3.

<p>Structure of 1α,25(OH)₂D₃ compared to the 2-methylene-19-nor analogs, 2MbisP and CAGE-3.</p

Effect of 1, 2, 3, 5, or 7 doses of 2MbisP, CAGE-3 or atRA on AREG, EPGN, EREG, and HB-EGF mRNA expression in skin.

<p>AREG, EPGN, EREG and HB-EGF mRNAs were analyzed by RT-PCR in skin taken 4 h after receiving the final dose of vehicle, 2MbisP (690 nmol/kg), CAGE-3 (0.25 nmol/kg), or atRA (224 nmol/kg). The data are expressed relative to the vehicle-treated group (treatment/vehicle). Significant differences from the vehicle group at each respective dose are indicated by an asterisk, *<i>P</i>≤0.05 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188887#pone.0188887.s005" target="_blank">S5 Table</a>).</p

Effect of 2MbisP and atRA on utricle size and epidermal thickness after 2, 7 and 21 topical doses.

<p>(A) Utricle area and (B) epidermal thickness was measured in H&E stained tissue sections taken from mice receiving vehicle, 2MbisP (690 nmol/kg), or atRA (224 nmol/kg). The data are expressed as percent of vehicle. Significant differences from the vehicle group at each dosing time point are indicated by an asterisk, *<i>P</i>≤0.05 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188887#pone.0188887.s001" target="_blank">S1 Table</a>).</p

Effect of 1, 2, 3, 5, or 7 doses of 2MbisP, CAGE-3 or atRA on utricle area and epidermal thickness.

<p>(A) Utricle area and (B) epidermal thickness were measured in stained tissue sections after the application of vehicle, 2MbisP (690 nmol/kg), CAGE-3 (0.25 nmol/kg body weight), or atRA (224 nmol/kg), and data are expressed as percent of vehicle. Significant differences from the vehicle group at each dosing time point are indicated by an asterisk, *<i>P</i>≤0.05 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188887#pone.0188887.s003" target="_blank">S3 Table</a>).</p

qPCR primers.

<p>qPCR primers.</p

Differential activity of 2-methylene-19-nor vitamin D analogs on growth factor gene expression in rhino mouse skin and comparison to all-<i>trans</i> retinoic acid

<div><p>While all 2-methylene-19-nor analogs of 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃) tested produce an increase in epidermal thickness in the rhino mouse, only a subset reduce utricle size (comedolysis). All-<i>trans</i> retinoic acid (atRA) also causes epidermal thickening and a reduction in utricle size in the rhino mouse. We now report that 2-methylene-19-nor-(20S)-1α-hydroxybishomopregnacalciferol (2MbisP), a comedolytic analog, increases epidermal thickening more rapidly than does atRA, while both reduce utricle area at an equal rate. Whereas unlike atRA, 2MbisP does not alter the epidermal growth factor receptor ligand, heparin-binding epidermal growth factor-like growth factor, it does increase the expression of both amphiregulin and epigen mRNA, even after a single dose. <i>In situ</i> hybridization reveals an increase in these transcripts throughout the closing utricle as well as in the interfollicular epidermis. The mRNAs for other EGFR ligands including betacellulin and transforming growth factor-α, as well as the epidermal growth factor receptor are largely unaffected by 2MbisP. Another analog, 2-methylene-19-nor-(20S)-26,27-dimethylene-1α,25-dihydroxyvitamin D3 (CAGE-3), produces epidermal thickening but fails to reduce utricle size or increase AREG mRNA levels. CAGE-3 modestly increases epigen mRNA levels, but only after 5 days of dosing. Thus, 2-MbisP produces unique changes in epidermal growth factor receptor ligand mRNAs that may be responsible for both epidermal proliferation and a reduction in utricle size.</p></div

<i>In situ</i> hybridization of AREG, EPGN, and HB-EGF mRNA skin treated with vehicle, 2MbisP, CAGE-3 or atRA.

<p>Compound or vehicle was applied topically every 24 h, followed by harvesting of the skin 4 h after the third dose. mRNA was detected in paraformaldehyde-fixed tissue sections using RNAscope probes and amplification system, followed by incubation with the substrate, diaminobenzidine, yielding a brown product. Tissue sections were counterstained with methyl green and were analyzed using brightfield microscopy. Bar = 0.1 mm.</p