Thyroxine differentially modulates the peripheral clock: lessons from the human hair follicle

Abstract

The human hair follicle (HF) exhibits peripheral clock activity, with knock-down of clock genes (BMAL1 and PER1) prolonging active hair growth (anagen) and increasing pigmentation. Similarly, thyroid hormones prolong anagen and stimulate pigmentation in cultured human HFs. In addition they are recognized as key regulators of the central clock that controls circadian rhythmicity. Therefore, we asked whether thyroxine (T4) also influences peripheral clock activity in the human HF. Over 24 hours we found a significant reduction in protein levels of BMAL1 and PER1, with their transcript levels also decreasing significantly. Furthermore, while all clock genes maintained their rhythmicity in both the control and T4 treated HFs, there was a significant reduction in the amplitude of BMAL1 and PER1 in T4 (100 nM) treated HFs. Accompanying this, cell-cycle progression marker Cyclin D1 was also assessed appearing to show an induced circadian rhythmicity by T4 however, this was not significant. Contrary to short term cultures, after 6 days, transcript and/or protein levels of all core clock genes (BMAL1, PER1, clock, CRY1, CRY2) were up-regulated in T4 treated HFs. BMAL1 and PER1 mRNA was also up-regulated in the HF bulge, the location of HF epithelial stem cells. Together this provides the first direct evidence that T4 modulates the expression of the peripheral molecular clock. Thus, patients with thyroid dysfunction may also show a disordered peripheral clock, which raises the possibility that short term, pulsatile treatment with T4 might permit one to modulate circadian activity in peripheral tissues as a target to treat clock-related disease.

Fig 1. BMAL1 and PER1 protein and transcript levels are reduced by thyroxine treatment.

HFs treated with T4 for 24 hours were stained for either BMAL1 or PER1 and transcript levels were assessed. (a) qRT-PCR demonstrated that CLOCK, BMAL1 and PER1, were significantly down regulated by T4 expression (24 hours). Protein expression was also assessed and quantified using immunohistomorphometry. (b,d) PER1 protein was significantly decreased by T4 at 6, 12,18 and 24 hours. BMAL1 protein levels were also decreased significantly by T4 at time points 6,12 and 18 hours showing a tendency to decrease at 0 and 24 hours. (mean (SD) * p < 0.05, *** p < 0.001, Student’s Ttest mean, results were pooled from multiple HFs from 3 patients). (scale bar = 50μm)

Fig 2. Thyroxine dampens clock gene expression over 48 hours.

To assess the influence of T4 of on the circadian expression of CLOCK, BMAL1, PER1 and Cyclin D1, HFs were synchronised, treated with T4 and sampled every 6 hours for 48 hours. (a, b, c & e) Quantitative-RT-PCR of clock transcripts showed that whilst both control and T4 treated HFs had rhythmic clock gene expression, which was supported by the confidence p values produced by the JTK cycle algorithm (e); it was reduced by T4 treatment. This was quantified by comparing the estimated amplitude measurement which demonstrated that the reduction in amplitude by T4 was significant for BMAL1 and PER1 (f). (d) Key cell cycle progression marker Cyclin D1 did not show a circadian expression pattern in the control group however, this appeared to be induced qualitatively by T4. (e) However this was not significant. (Mann-Whitney, * p < 0.05, mean (SD), HFs were pooled data from 4 donors).

Fig 3. Clock transcript levels and PER1 protein levels are increased after 6 days treatment with T4.

The effects of T4 on clock gene and protein expression were assessed on HFs treated for 6 days by immunofluorescence and qRT-PCR. (a) All core clock genes were significantly up-regulated at 6 days by T4 treatment. The Protein expression of PER1 was also up-regulated after 6 days (b & c) however, BMAL1 expression remained unchanged (b & D). (Mann-Whitney, * p < 0.05, mean (SD), HFs were pooled data from 3 patients). (scale bar = 50μm)

Fig 4. K15+ stem cells express BMAL1 and PER1 protein.

(a) After establishing that K15 positive cells express BMAL1 and PER1 the role T4 on clock gene expression in the k15+ bulge stem cells was assessed by qRT-PCR and quantitative immunohistomorphometry. Transcript levels of BMAL1 and PER1 were upregulated after 6 days (b), (c-e) however, whilst PER1 showed a tendency to increase, neither BMAL1 or PER1 protein levels increased significantly. (scale bar = 50 μm, HFs were pooled from three donors, p < 0.05 *, ** p < 0.01, mean (SD)).