Tatyana Y. Sharova

P63 REGULATES SATB1 TO CONTROL TISSUE-SPECIFIC CHROMATIN REMODELING DURING DEVELOPMENT OF THE EPIDERMIS

Genome organizer Satb1 is regulated by p63 and contributes to epidermal morphogenesis by remodeling chromatin structure and gene expression at the epidermal differentiation complex locus.

Bone morphogenetic protein signaling regulates the size of hair follicles and modulates the expression of cell cycle-associated genes

Bone morphogenetic protein (BMP) signaling is involved in the regulation of a large variety of developmental programs, including those controlling organ sizes. Here, we show that transgenic (TG) mice overexpressing the BMP antagonist noggin (promoter, K5) are characterized by a marked increase in size of anagen hair follicles (HFs) and by the replacement of zig-zag and auchen hairs by awl-like hairs, compared with the age-matched WT controls. Markedly enlarged anagen HFs of TG mice show increased proliferation in the matrix and an increased number of hair cortex and medulla cells compared with WT HFs. Microarray and real-time PCR analyses of the laser-captured hair matrix cells show a strong decrease in expression of Cdk inhibitor p27Kip1 and increased expression of selected cyclins in TG vs. WT mice. Similar to TG mice, p27Kip1 knockout mice also show an increased size of anagen HFs associated with increased cell proliferation in the hair bulb. Primary epidermal keratinocytes (KC) from TG mice exhibit significantly increased proliferation and decreased p27Kip1 expression, compared with WT KC. Alternatively, activation of BMP signaling in HaCaT KC induces growth arrest, stimulates p27Kip1 expression, and positively regulates p27Kip1 promoter activity, thus further supporting a role of p27Kip1 in mediating the effects of BMP signaling on HF size. These data suggest that BMP signaling plays an important role in regulating cell proliferation and controls the size of anagen HFs by modulating the expression of cell-cycle-associated genes in hair matrix KC.

Lhx2 differentially regulates Sox9, Tcf4 and Lgr5 in hair follicle stem cells to promote epidermal regeneration after injury

The Lhx2 transcription factor plays essential roles in morphogenesis and patterning of ectodermal derivatives as well as in controlling stem cell activity. Here, we show that during murine skin morphogenesis, Lhx2 is expressed in the hair follicle (HF) buds, whereas in postnatal telogen HFs Lhx2+ cells reside in the stem cell-enriched epithelial compartments (bulge, secondary hair germ) and co-express selected stem cell markers (Sox9, Tcf4 and Lgr5). Remarkably, Lhx2+ cells represent the vast majority of cells in the bulge and secondary hair germ that proliferate in response to skin injury. This is functionally important, as wound re-epithelization is significantly retarded in heterozygous Lhx2 knockout (+/–) mice, whereas anagen onset in the HFs located closely to the wound is accelerated compared with wild-type mice. Cell proliferation in the bulge and the number of Sox9+ and Tcf4+ cells in the HFs closely adjacent to the wound in Lhx2+/– mice are decreased in comparison with wild-type controls, whereas expression of Lgr5 and cell proliferation in the secondary hair germ are increased. Furthermore, acceleration of wound-induced anagen development in Lhx2+/– mice is inhibited by administration of Lgr5 siRNA. Finally, Chip-on-chip/ChIP-qPCR and reporter assay analyses identified Sox9, Tcf4 and Lgr5 as direct Lhx2 targets in keratinocytes. These data strongly suggest that Lhx2 positively regulates Sox9 and Tcf4 in the bulge cells, and promotes wound re-epithelization, whereas it simultaneously negatively regulates Lgr5 in the secondary hair germ and inhibits HF cycling. Thus, Lhx2 operates as an important regulator of epithelial stem cell activity in the skin response to injury.

Noggin overexpression inhibits eyelid opening by altering epidermal apoptosis and differentiation

Contact of developing sensory organs with the external environment is established via the formation of openings in the skin. During eye development, eyelids first grow, fuse and finally reopen, thus providing access for visual information to the retina. Here, we show that eyelid opening is strongly inhibited in transgenic mice overexpressing the bone morphogenetic protein (BMP) antagonist noggin from the keratin 5 (K5) promoter in the epidermis. In wild‐type mice, enhanced expression of the kinase‐inactive form of BMPR‐IB mediated by an adenovirus vector also inhibits eyelid opening. Noggin overexpression leads to reduction of apoptosis and retardation of cell differentiation in the eyelid epithelium, which is associated with downregulation of expression of the apoptotic receptors (Fas, p55 kDa TNFR), Id3 protein and keratinocyte differentiation markers (loricrin, involucrin). BMP‐4, but not EGF or TGF‐α, accelerates opening of the eyelid explants isolated from K5‐Noggin transgenic mice when cultured ex vivo. These data suggest that the BMP signaling pathway plays an important role in regulation of genetic programs of eyelid opening and skin remodeling during the final steps of eye morphogenesis.

p63 and Brg1 control developmentally regulated higher-order chromatin remodelling at the epidermal differentiation complex locus in epidermal progenitor cells

Chromatin structural states and their remodelling, including higher-order chromatin folding and three-dimensional (3D) genome organisation, play an important role in the control of gene expression. The role of 3D genome organisation in the control and execution of lineage-specific transcription programmes during the development and differentiation of multipotent stem cells into specialised cell types remains poorly understood. Here, we show that substantial remodelling of the higher-order chromatin structure of the epidermal differentiation complex (EDC), a keratinocyte lineage-specific gene locus on mouse chromosome 3, occurs during epidermal morphogenesis. During epidermal development, the locus relocates away from the nuclear periphery towards the nuclear interior into a compartment enriched in SC35-positive nuclear speckles. Relocation of the EDC locus occurs prior to the full activation of EDC genes involved in controlling terminal keratinocyte differentiation and is a lineage-specific, developmentally regulated event controlled by transcription factor p63, a master regulator of epidermal development. We also show that, in epidermal progenitor cells, p63 directly regulates the expression of the ATP-dependent chromatin remodeller Brg1, which binds to distinct domains within the EDC and is required for relocation of the EDC towards the nuclear interior. Furthermore, Brg1 also regulates gene expression within the EDC locus during epidermal morphogenesis. Thus, p63 and its direct target Brg1 play an essential role in remodelling the higher-order chromatin structure of the EDC and in the specific positioning of this locus within the landscape of the 3D nuclear space, as required for the efficient expression of EDC genes in epidermal progenitor cells during skin development.

Bone morphogenetic protein antagonist noggin promotes skin tumorigenesis via stimulation of the Wnt and Shh signaling pathways

Bone morphogenetic proteins (BMPs) play pivotal roles in the regulation of skin development. To study the role of BMPs in skin tumorigenesis, BMP antagonist noggin was used to generate keratin 14-targeted transgenic mice. In contrast to wild-type mice, transgenic mice developed spontaneous hair follicle-derived tumors, which resemble human trichofolliculoma. Global gene expression profiles revealed that in contrast to anagen hair follicles of wild-type mice, tumors of transgenic mice showed stage-dependent increases in the expression of genes encoding the selected components of Wnt and Shh pathways. Specifically, expression of the Wnt ligands increased at the initiation stage of tumor formation, whereas expression of the Wnt antagonist and tumor suppressor Wnt inhibitory factor-1 decreased, as compared with fully developed tumors. In contrast, expression of the components of Shh pathway increased in fully developed tumors, as compared with the tumor placodes. Consistent with the expression data, pharmacological treatment of transgenic mice with Wnt and Shh antagonists resulted in the stage-dependent inhibition of tumor initiation, and progression, respectively. Furthermore, BMP signaling stimulated Wnt inhibitory factor-1 expression and promoter activity in cultured tumor cells and HaCaT keratinocytes, as well as inhibited Shh expression, as compared with the corresponding controls. Thus, tumor suppressor activity of the BMPs in skin epithelium depends on the local concentrations of noggin and is mediated at least in part via stage-dependent antagonizing of Wnt and Shh signaling pathways.

Fas Signaling Is Involved in the Control of Hair Follicle Response to Chemotherapy

Chemotherapeutic agents induce p53-dependent apoptosis in the hair follicle (HF) resulting in hair loss, a common side effect of cancer therapy. Here, we show that Fas as a p53 target plays important role in the HF response to cyclophosphamide. Specifically, we demonstrate that Fas is up-regulated in HF keratinocytes after cyclophosphamide treatment, Fas ligand–neutralizing antibody partially inhibits HF response to cyclophosphamide in wild-type mice, and Fas knockout mice show significant retardation of cyclophosphamide-induced HF involution associated with reduced Fas-associated death domain and caspase-8 expression. These data raise a possibility to explore blockade of Fas signaling as a part of complex local therapy for inhibiting keratinocyte apoptosis and hair loss induced by chemotherapy.

Bone morphogenetic protein signaling suppresses wound-induced skin repair by inhibiting keratinocyte proliferation and migration.

Bone morphogenetic protein (BMP) signalling plays a key role in the control of skin development and postnatal remodelling by regulating keratinocyte proliferation, differentiation and apoptosis. To study the role of BMPs in wound-induced epidermal repair, we used transgenic mice overexpressing the BMP downstream component Smad1 under the control of a K14 promoter as an in vivo model, as well as ex vivo and in vitro assays. K14-caSmad1 mice exhibited retarded wound healing associated with significant inhibition of proliferation and increased apoptosis in healing wound epithelium. Furthermore, microarray and qRT-PCR analyses revealed decreased expression of a number of cytoskeletal/cell motility-associated genes including wound-associated keratins (Krt16, Krt17) and Myo5a, in the epidermis of K14-caSmad1 mice versus wild-type controls during wound healing. BMP treatment significantly inhibited keratinocyte migration ex vivo, and primary keratinocytes of K14-caSmad1 mice showed retarded migration compared to wild-type controls. Finally, siRNA-mediated silencing of Bmpr-1B in primary mouse keratinocytes accelerated cell migration and was associated with increased expression of Krt16, Krt17 and Myo5a compared to controls. Thus, this study demonstrates that BMPs inhibit keratinocyte proliferation, cytoskeletal organization and migration in regenerating skin epithelium during wound healing, and raises a possibility for using BMP antagonists for the management of chronic wounds.

Matrix Metalloproteinase-9 Is Involved in the Regulation of Hair Canal Formation

To the Editor: Hair follicle (HF) morphogenesis is governed by a series of signals exchanged between the epidermal keratinocytes committed to HF-specific differentiation and the mesenchymal cells forming the follicular papilla (Fuchs, 2007; Schmidt-Ullrich and Paus, 2005). These interactions lead to the construction of the hair bulb, in which keratinocytes rapidly proliferate and differentiate into several cell populations forming the medulla, cortex and cuticle of the hair shaft, as well as the cuticle, Huxley and Henle layers of the inner root sheath. During the final steps of development, the HF elongates up to its maximal length and the differentiating hair shaft emerges through the epidermis via the hair canal that is formed at the distal portion of the HF epithelium (Schmidt-Ullrich and Paus, 2005). Morphologically, the hair or pilary canal is considered as a continuation of the hair germ upward into the epidermis and its formation begins at stage 3 of HF morphogenesis, when the epidermal cells above the HF become oriented perpendicularly to the surface of the skin (Pinkus, 1958). During later stages of HF development (stage 4–6), these cells keratinized and form a central core surrounded by a tubular layer of epithelial cells inside of the epidermis and HF infundibulum (Hashimoto, 1970). Finally, the centre of the keratinized tissue in a lumen of the hair canal is eliminated by the tip of the emerging hair (stages 7–8 of development) (Breathnach, 1971; Sengel, 1976). Despite recent advances in identification of the molecular signaling/transcription networks that govern the development of the skin and HF (Fuchs, 2007), the mechanisms controlling the epithelial tissue remodeling during hair canal formation remain unclear. It has been long appreciated that matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) are involved in tissue remodeling during embryonic and postnatal development (Ortega et al., 2003; Werb and Chin, 1998). MMP-9 (gelatinase B) is involved in the degradation of extracellular matrix and/or modulation of growth factor signaling through its degrading activity during development (Mott and Werb, 2004). MMP-9 activity is present in the culture medium containing explants of developing HFs (Obana et al., 1996), as well as in normal adult mouse skin with actively growing (anagen) HFs (Paus et al., 1994). In human anagen HFs, MMP-9 is expressed in the Henle’s layer of the inner root sheath (Jarrousse et al., 2001). To elucidate a role for MMP-9 in the control of HF development and hair canal formation, its expression was studied by immunohistochemistry in cryosections of embryonic and postnatal skin of C57BL/6 mice (Botchkarev et al., 2001; Botchkarev et al., 1999; Sharov et al., 2003). Immunohistochamical data were correlated to the micro-anatomy of the developing hair canal depicted by high-resolution light microscopy (HRLM), as described previously (Magerl et al., 2001). MMP-9 expression was not seen in the epidermis and developing HF placodes (Fig. 1 A, B). Starting from E17.5, single MMP-9 expressing cells became visible in the central part of the developing intra-epidermal hair canal (stage 3 of HF morphogenesis; Fig. 1 C, D). In stage 4–6 HFs, the number of MMP-9 expressing cells increased in central portion of the hair canal, which walls showed appearance of the trichohyalin granules, while the other HF compartments (hair bulb, sebaceous gland, connective tissue sheath) did not show any detectable MMP-9 immunoreactivity (Fig. 1 E, F). During final stages of HF development (stage 7 and 8), MMP9+ cells disappeared from the hair canal and were visible only in the dermis (Fig. 1 G, H). Figure 1 MMP-9 is expressed in the hair canal of developing hair follicles To further define the functions of MMP-9 in hair canal formation, the dynamics of HF development were compared between wild-type (n=5) and MMP-9 knockout (−/−) mice (n=7) at postnatal day 4.5 (P4.5). It was shown previously that MMP-9 knockout mice are characterized by the defects in ossification and angiogenesis, delayed wound healing, and are resistant to experimentally-induced bullous pemphigoid (Kyriakides et al., 2009; Liu et al., 1998; Vu et al., 1998). Consistently with the expression pattern for MMP-9 in the developing skin (Fig. 1), histo-morphometric analyses of the early postnatal skin showed significant (p<0.05) retardation of the process of hair shaft emergence through the epidermis in MMP-9 −/− mice versus wild-type controls (Fig. 2 A). Despite apparently normal earlier stages of HF development and skin morphology, MMP-9 −/− mice showed significant (p<0.05) increase in a number of HFs at stage 7 of morphogenesis in which hair shaft was retained inside of the hair canal versus the age-matched wild-type mice that showed the increased number of stage 8 HFs and more rapid hair shaft appearance at the skin surface (Fig. 2 B, C). In addition to these differences, the width of the hair canal determined as a distance between the internal surfaces of its walls and used as an important parameter of the dynamics of its formation was significantly (p<0.05) decreased in MMP9 −/− mice compared to wild-type mice (Fig. 2 D–F). Figure 2 Retardation of the hair shaft emergence through the epidermis in MMP-9 knockout mice These data suggest that MMP-9 plays an important role in the extracellular matrix remodelling during hair canal formation. MMP-9 degrades the denatured collagens (gelatin), as well as can cleave native type IV, V and XI collagens, elastin and a number of other molecules (Nothnick, 2008). MMP-9 is expressed in the central part of the hair canal (Fig. 1 D, F) containing keratinized “core cells”, which later desquamate and form the initial lumen of the hair canal prior to the appearance of the hair tip (Hashimoto, 1970). By active extracellular matrix remodeling, MMP-9 may promote the formation of the initial lumen, thus preparing the hair canal for the hair shaft emergence and preventing its walls from a damage induced by the growing hair fiber. However, it is unclear whether MMP-9 and/or other metalloproteinases play a role in the control of hair canal formation during morphogenesis of the human HFs. Furthermore, mechanisms controlling the expression of MMP-9 during hair canal formation in mice also remain unclear. In other cell types (lymphocytes, fibroblasts, osteoblasts, isolated keratinocytes, etc.), MMP-9 expression is regulated by the Wnt, TGF-beta/BMP, HGF and EGF signaling pathways (McCawley et al., 1998; Nothnick, 2008; Wu et al., 2007). These signaling pathways also serve as key regulators of HF development (Fuchs, 2007; Schmidt-Ullrich and Paus, 2005), which suggest a possibility for their involvement in the control of MMP-9 expression in a subset of keratinocytes that specify the hair canal areas in the epidermis. Future research in this direction will help in further understanding the roles of MMP-9 and other metalloproteinases in the control of extracellular matrix remodeling in the developing skin, as well as in establishing the molecular signaling networks that underlie the complex programs of skin appendage morphogenesis.

Complex changes in the apoptotic and cell differentiation programs during initiation of the hair follicle response to chemotherapy

Chemotherapy has severe side-effects for normal rapidly proliferating organs, such as hair follicle, and causes massive apoptosis in hair matrix keratinocytes followed by hair loss. To define the molecular signature of hair follicle response to chemotherapy, human scalp hair follicles cultured ex vivo were treated with doxorubicin and global microarray analysis was performed 3 hours after treatment. Microarray data revealed changes in expression of 504 genes in doxorubicin-treated hair follicles versus the controls. Among these genes, upregulations of several tumor necrosis factor family of apoptotic receptors (FAS, TRAIL receptors 1/2), as well as of a large number of the keratin-associated protein genes were seen after doxorubicin treatment. Hair follicle apoptosis induced by doxorubicin was significantly inhibited by either TRAIL neutralizing antibody or caspase 8 inhibitor, thus suggesting a novel role for TRAIL receptor signaling in mediating doxorubicin-induced hair loss. These data demonstrate that the early phase of the hair follicle response to doxorubicin includes upregulation of apoptosis-associated markers, as well as substantial re-organization of the terminal differentiation programs in hair follicle keratinocytes. These data provide an important platform for further studies towards the design of novel approaches for management of chemotherapy-induced hair loss.