Similarities in the role of FoxE1 in thyrocytes and keratinocytes?
M Bullock1, M Jehani2, P Bowden2 & M Ludgate1
Patients with non-functional FOXE1 (forkhead transcription factor) display congenital hypothyroidism, spikey hair and other abnormalities. In the thyroid, FoxE1 controls migration of the developing gland and adult expression of thyroid specific genes. Our earlier studies demonstrated FOXE1 protein expression in keratinocytes of the epidermis and hair-follicle outer root sheath.
We aimed to characterise the expression and functional activity of FOXE1 during proliferation and differentiation of HaCaT, a spontaneously immortalised human epidermal keratinocyte cell-line.
Keratinocytes cultured in low Ca2+ proliferate, elevating the Ca2+ concentration above 1.0 mM induces differentiation. HaCaT cells were plated in 0.06 or 0.15 mM Ca2+ and increased to 1.8mM when they reached 80% confluence. Cultures were maintained for a further 6 days. mRNA was extracted at various time points, reverse transcribed and transcripts for FOXE1 and the APRT housekeeper gene measured using SYBR green and a Stratagene MX3000. Variations in FOXE1 functional activity were measured in a subclone of HaCaT cells that expresses a FOXE1 responsive luciferase reporter.
Even in low Ca2+ medium, cell contact with increasing confluence induced differentiation and a 2-fold increase in FOXE1 transcripts. The calcium-shift produced a transient dip in FOXE1 transcripts which then continued to rise achieving levels 3 to 5 fold higher than at the outset, before finally declining. This was accompanied by temporal expression of differentiation markers, keratin 10 and filaggrin, evaluated by IMF. Similar results were obtained at both plating Ca2+ concentrations and in the HaCaT subclone, which also displayed a 3-fold increase in luciferase. Subsequent studies revealed that TIMP-3 (promoter contains FoxE1 consensus) transcripts were upregulated 4 to 5 fold overall and also displayed a temporal reduction after adding Ca2+.
FoxE1 transcripts and activity increase with keratinocyte differentiation and target at least one gene implicated in modification of the extra-cellular matrix, a situation of potential relevance to hair-follicle migration.