Desmoglein 4 is regulated by transcription factors implicated in hair shaft differentiation

Abstract

The hair fiber is made of specialized keratinocytes, known as trichocytes, that primarily express hair keratins, which are cemented by a multitude of keratin-associated proteins (KAPs). The hair keratins form the intermediate filament cytoskeleton of the trichocytes, which are linked to abundant cell-cell adhesion junctions, called desmosomes. Desmoglein 4 (DSG4) is the major desmosomal cadherin expressed in the hair shaft cortex where the hair keratins are highly expressed. In humans, mutations affecting either the hair keratins or DSG4 lead to beaded hair phenotypes with features of monilethrix. In this work, we postulated that the regulatory pathways governing the expression of hair shaft components, such as hair keratins and DSG4, are shared. Therefore, we studied the transcriptional regulation of DSG4 by transcription factors/pathways that are known regulators of hair keratin or KAP expression. We show that HOXC13, LEF1 and FOXN1 repress DSG4 transcription and provide in vitro and in vivo evidence correlating the Notch pathway with the activation and/or maintenance of DSG4 expression in the hair follicle.

Figure 1

Dane and Herman staining of a normal human HF (A) and a Dsg4 −/− rat Vibrissae follicle (B). The dermal papilla is light blue. Note the abnormal keratinized mass at the tip of the abnormal precortex in (B) which is a hallmark of Dsg4 mutations in rodents. Abbreviations: dermal papilla, DP; Outer root sheath, ORS; Inner root sheath, IRS; Cortex, Co; Pre-cortex, pre-Co; Cuticle, Cu. Scale bars: A, 100 µm; B, 40 µm.

Figure 2

Dsg4 is expressed in the cortex and IRS cuticle of rat vibrissae follicles and rat and mouse pelage follicles during anagen but absent from Dsg4−/− follicles. (A–L) Dsg4 is in green, the cortex hair keratin Ha1 is in red (B,E,G,I,K), the IRS keratin IRS3a.1 is in red (C,D,F,H,J,L), DNA counterstain DAPI is in blue. (A–D) Rat vibrissae follicles. (E–H) Rat pelage follicles. (I–L) mouse pelage follicles. The green staining in the dermis is background staining since the anti-Dsg4 is mouse monoclonal antibody used on mouse tissue. White dotted line in this and subsequent figures represents the basement membrane region. Scale bars: A–D, 800 µm; E–H, 100 µm; I–L, 150 µm.

Figure 2

Dsg4 is expressed in the cortex and IRS cuticle of rat vibrissae follicles and rat and mouse pelage follicles during anagen but absent from Dsg4−/− follicles. (A–L) Dsg4 is in green, the cortex hair keratin Ha1 is in red (B,E,G,I,K), the IRS keratin IRS3a.1 is in red (C,D,F,H,J,L), DNA counterstain DAPI is in blue. (A–D) Rat vibrissae follicles. (E–H) Rat pelage follicles. (I–L) mouse pelage follicles. The green staining in the dermis is background staining since the anti-Dsg4 is mouse monoclonal antibody used on mouse tissue. White dotted line in this and subsequent figures represents the basement membrane region. Scale bars: A–D, 800 µm; E–H, 100 µm; I–L, 150 µm.

Figure 2

Dsg4 is expressed in the cortex and IRS cuticle of rat vibrissae follicles and rat and mouse pelage follicles during anagen but absent from Dsg4−/− follicles. (A–L) Dsg4 is in green, the cortex hair keratin Ha1 is in red (B,E,G,I,K), the IRS keratin IRS3a.1 is in red (C,D,F,H,J,L), DNA counterstain DAPI is in blue. (A–D) Rat vibrissae follicles. (E–H) Rat pelage follicles. (I–L) mouse pelage follicles. The green staining in the dermis is background staining since the anti-Dsg4 is mouse monoclonal antibody used on mouse tissue. White dotted line in this and subsequent figures represents the basement membrane region. Scale bars: A–D, 800 µm; E–H, 100 µm; I–L, 150 µm.

Figure 3

Dsg4 is expressed in the forming cortex on day 1 (d1) and is more prominent in the cortex in mid to late anagen (d11) in rat pelage follicles. Dsg4 is still expressed in the receding hair cortex and around the forming club hair during catagen on d19 in rat pelage follicles. Cortical staining of Dsg4 is also evident in mid anagen mouse pelage follicles on d7 (see insets). Scale bars: Rat, 70 µm, Mouse, 50 µm.

Figure 4

DSG4 expression (green) overlaps with that of HOXC13 (A,B), FOXN1 (C,D), and LEF1 (all is red) in the precortex and sometimes cortex of the human hair follicles. Note that DSG4 and HOXC13 are co-expressed also in the IRS cuticle (arrows in B) and that DSG4 and FOXN1 overlap in the differentiating cortex (D). DAPI counterstain is in blue. Scale bars: A–E, 200 µm; B–F, 80 µm.

Figure 5

The endogenous activity of Dsg4 upstream region reporter constructs is repressed by some transcription factors in HaCaT keratinocytes. (A) ~3Kb and ~1kb upstream of human (H1,3) and mouse (m1,3) show endogenous reporter construct activity in HaCaT keratinocytes compared to the pGL3 basic vector control. (B) HOXC13, but not a mutant version (Mut HOXC13) that is not capable of binding DNA, also represses H3 in the same cells. (C) FOXN1, but not MSX2, also represses H3 although to a lesser extent than HOXC13 and LEF1. (D) LEF1 represses H3 activity in HaCaT keratinocytes to background level (also H1, m3 and m1, not shown). Error bars, standard deviation in figures 5 and 6. Student t-test was used to assess significance between control and treated groups with a p-value cutoff of 0.05 in figures 5 and 6.

Figure 5

The endogenous activity of Dsg4 upstream region reporter constructs is repressed by some transcription factors in HaCaT keratinocytes. (A) ~3Kb and ~1kb upstream of human (H1,3) and mouse (m1,3) show endogenous reporter construct activity in HaCaT keratinocytes compared to the pGL3 basic vector control. (B) HOXC13, but not a mutant version (Mut HOXC13) that is not capable of binding DNA, also represses H3 in the same cells. (C) FOXN1, but not MSX2, also represses H3 although to a lesser extent than HOXC13 and LEF1. (D) LEF1 represses H3 activity in HaCaT keratinocytes to background level (also H1, m3 and m1, not shown). Error bars, standard deviation in figures 5 and 6. Student t-test was used to assess significance between control and treated groups with a p-value cutoff of 0.05 in figures 5 and 6.

Figure 5

The endogenous activity of Dsg4 upstream region reporter constructs is repressed by some transcription factors in HaCaT keratinocytes. (A) ~3Kb and ~1kb upstream of human (H1,3) and mouse (m1,3) show endogenous reporter construct activity in HaCaT keratinocytes compared to the pGL3 basic vector control. (B) HOXC13, but not a mutant version (Mut HOXC13) that is not capable of binding DNA, also represses H3 in the same cells. (C) FOXN1, but not MSX2, also represses H3 although to a lesser extent than HOXC13 and LEF1. (D) LEF1 represses H3 activity in HaCaT keratinocytes to background level (also H1, m3 and m1, not shown). Error bars, standard deviation in figures 5 and 6. Student t-test was used to assess significance between control and treated groups with a p-value cutoff of 0.05 in figures 5 and 6.

Figure 5

The endogenous activity of Dsg4 upstream region reporter constructs is repressed by some transcription factors in HaCaT keratinocytes. (A) ~3Kb and ~1kb upstream of human (H1,3) and mouse (m1,3) show endogenous reporter construct activity in HaCaT keratinocytes compared to the pGL3 basic vector control. (B) HOXC13, but not a mutant version (Mut HOXC13) that is not capable of binding DNA, also represses H3 in the same cells. (C) FOXN1, but not MSX2, also represses H3 although to a lesser extent than HOXC13 and LEF1. (D) LEF1 represses H3 activity in HaCaT keratinocytes to background level (also H1, m3 and m1, not shown). Error bars, standard deviation in figures 5 and 6. Student t-test was used to assess significance between control and treated groups with a p-value cutoff of 0.05 in figures 5 and 6.

Figure 6

Notch is a potential activator of Dsg4 expression in vitro and in vivo. (A) NICD, the downstream effector of Notch signaling, and Hoxc12 increase H3 reporter construct activity in HaCaT keratinocytes. (B–D) The precortical abnormal mass of keratinization is common to lah (arrow in C) and PSDCKO (arrow in D and F) mutant follicles compared to WT follicles (B). (E,F) Dsg4 expression (green) is markedly downregulated in PSDCKO follicles (F) compared to normal HFs (E). DAPI counterstain is in blue. Scale bars: B–F, 40µm.

Figure 6

Notch is a potential activator of Dsg4 expression in vitro and in vivo. (A) NICD, the downstream effector of Notch signaling, and Hoxc12 increase H3 reporter construct activity in HaCaT keratinocytes. (B–D) The precortical abnormal mass of keratinization is common to lah (arrow in C) and PSDCKO (arrow in D and F) mutant follicles compared to WT follicles (B). (E,F) Dsg4 expression (green) is markedly downregulated in PSDCKO follicles (F) compared to normal HFs (E). DAPI counterstain is in blue. Scale bars: B–F, 40µm.