Proteomic analysis of differentially expressed skin proteins in iRhom2Uncv mice

A mouse homozygous for the spontaneous mutation uncovered (Uncv) has a hairless phenotype. A 309-bp non-frameshift deletion mutation in the N-terminal cytoplasmic domain of iRhom2 was identified in Uncv mice (iRhom2^Uncv) using target region sequencing. The detailed molecular basis for how the iRhom2 mutation causes the hairless phenotype observed in the homozygous iRhom2^Uncv mouse remains unknown. To identify differentially expressed proteins in the skin of wild-type and homozygous iRhom2^Uncv littermates at postnatal day 5, proteomic approaches, including two-dimensional gel electrophoresis and mass spectrometry were used. Twelve proteins were differentially expressed in the skin in a comparison between wild-type and homozygous iRhom2^Uncv mice. A selection of the proteomic results were tested and verified using qRT-PCR, western blot and immunohistochemistry. These data indicate that differentially expressed proteins, especially KRT73, MEMO1 and Coro-1, might participate in the mechanism by which iRhom2 regulates the development of murine skin. [BMB Reports 2015; 48(1): 19-24]     

Mammalian iRhoms have distinct physiological functions including an essential role in TACE regulation

Loss of iRhom2, a catalytically inactive rhomboid‐like protein, blocks maturation of TACE/ADAM17 in macrophages, resulting in defective shedding of the cytokine tumor necrosis factor. Apart from the resulting inflammatory defects, iRhom2‐null mice appear normal: they do not show the several defects seen in TACE knockouts, suggesting that TACE maturation is independent of iRhom2 in cells other than macrophages. Here we show that the physiological role of iRhoms is much broader. iRhom1 knockout mice die within 6 weeks of birth. They show a severe phenotype, with defects in several tissues including highly penetrant brain haemorrhages. The non‐overlapping phenotypes imply that iRhom 1 and 2 have distinct physiological roles, although at a cellular level both promote the maturation of TACE (but not other ADAM proteases). Both iRhoms are co‐expressed in many contexts where TACE acts. We conclude that all TACE activity, constitutive and regulated, requires iRhom function. iRhoms are therefore essential and specific regulators of TACE activity, but our evidence also implies that they must have additional physiologically important clients.     

LPA-producing enzyme PA-PLA₁α regulates hair follicle development by modulating EGFR signalling

Recent genetic studies of human hair disorders have suggested a critical role of lysophosphatidic acid (LPA) signalling in hair follicle development, mediated by an LPA-producing enzyme, phosphatidic acid-selective phospholipase A(1)α (PA-PLA(1)α, also known as LIPH), and a recently identified LPA receptor, P2Y5 (also known as LPA(6)). However, the underlying molecular mechanism is unknown. Here, we show that epidermal growth factor receptor (EGFR) signalling underlies LPA-induced hair follicle development. PA-PLA(1)α-deficient mice generated in this study exhibited wavy hairs due to the aberrant formation of the inner root sheath (IRS) in hair follicles, which resembled mutant mice defective in tumour necrosis factor α converting enzyme (TACE), transforming growth factor α (TGFα) and EGFR. PA-PLA(1)α was co-localized with TACE, TGFα and tyrosine-phosphorylated EGFR in the IRS. In PA-PLA(1)α-deficient hair follicles, cleaved TGFα and tyrosine-phosphorylated EGFR, as well as LPA, were significantly reduced. LPA, P2Y5 agonists and recombinant PA-PLA(1)α enzyme induced P2Y5- and TACE-mediated ectodomain shedding of TGFα through G12/13 pathway and consequent EGFR transactivation in vitro. These data demonstrate that a PA-PLA(1)α-LPA-P2Y5 axis regulates differentiation and maturation of hair follicles via a TACE-TGFα-EGFR pathway, thus underscoring the physiological importance of LPA-induced EGFR transactivation.     

Effects of Wnt-10b on hair shaft growth in hair follicle cultures

Wnts are deeply involved in the proliferation and differentiation of skin epithelial cells. We previously reported the differentiation of cultured primary skin epithelial cells toward hair shaft and inner root sheath (IRS) of the hair follicle via beta-catenin stabilization caused by Wnt-10b, however, the effects of Wnt-10b on cultured hair follicles have not been reported. In the present study, we examined the effects of Wnt-10b on shaft growth using organ cultures of whisker hair follicles in serum-free conditions. No hair shaft growth was observed in the absence of Wnt-10b, whereas its addition to the culture promoted elongation of the hair shaft, intensive incorporation of BrdU in matrix cells flanking the dermal papilla (DP), and beta-catenin stabilization in DP and IRS cells. These results suggest a promoting effect of Wnt-10b on hair shaft growth that is involved with stimulation of the DP via Wnt-10b/beta-catenin signalling, proliferation of matrix cells next to the DP, and differentiation of IRS cells by Wnt-10b.     

Expression of nucleosomal protein HMGN1 in the cycling mouse hair follicle

Here we examine the expression pattern of HMGN1, a nucleosome binding protein that affects chromatin structure and activity, in the hair follicle and test whether loss of HMGN1 affects the development or cycling of the follicle. We find that at the onset of hair follicle development, HMGN1 protein is expressed in the epidermal placode and in aggregated dermal fibroblasts. In the adult hair follicle, HMGN1 is specifically expressed in the basal layer of epidermis, in the outer root sheath, in the hair bulb, but not in the inner root sheath and hair shaft. The expression pattern of HMGN1 is very similar to p63, suggesting a role for HMGN1 in the transiently amplifying cells. We also find HMGN1 expression in some, but not all hair follicle stem cells as detected by its colocalization with Nestin and with BrdU label-retaining cells. The appearance of the skin and hair follicle of Hmgn1^?/? mice was indistinguishable from that of their Hmgn1^+/+ littermates. We found that in the hair follicle the expression of HMGN2 is very similar to HMGN1 suggesting functional redundancy between these closely related HMGN variants.     

Inhibition of BMP signaling in P-Cadherin positive hair progenitor cells leads to trichofolliculoma-like hair follicle neoplasias

Background  

Skin stem cells contribute to all three major lineages of epidermal appendages, i.e., the epidermis, the hair follicle, and the sebaceous gland. In hair follicles, highly proliferative committed progenitor cells, called matrix cells, are located at the base of the follicle in the hair bulb. The differentiation of these early progenitor cells leads to specification of a central hair shaft surrounded by an inner root sheath (IRS) and a companion layer. Multiple signaling molecules, including bone morphogenetic proteins (BMPs), have been implicated in this process.     

Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles

We investigated the expression of P2X₅, P2X₇, P2Y₁ and P2Y₂ receptor subtypes in adult human anagen hair follicles and in relation to markers of proliferation [proliferating cell nuclear antigen (PCNA) and Ki-67], keratinocyte differentiation (involucrin) and apoptosis (anticaspase-3). Using immunohistochemistry, we showed that P2X₅, P2Y₁ and P2Y₂ receptors were expressed in spatially distinct zones of the anagen hair follicle: P2Y₁ receptors in the outer root sheath and bulb, P2X₅ receptors in the inner and outer root sheaths and medulla and P2Y₂ receptors in living cells at the edge of the cortex/medulla. P2X₇ receptors were not expressed. Colocalisation experiments suggested different functional roles for these receptors: P2Y₁ receptors were associated with bulb and outer root sheath keratinocyte proliferation, P2X₅ receptors were associated with differentiation of cells of the medulla and inner root sheaths and P2Y₂ receptors were associated with early differentiated cells in the cortex/medulla that contribute to the formation of the hair shaft. The therapeutic potential of purinergic agonists and antagonists for controlling hair growth is discussed.     

The mutant gene wellhaarig disturbs the differentiation of hair follicle cells in the mouse]

First generation (G1) hairs in mice homozygous for the wellhaarig (we) gene are wavy and shorter than in normal mice; basal regions of the hairs are deformed. Follicles of G1 hairs in mid-dorsal region of 8-, 12- and 16-days old we/we mice were examined. Huxley cells of the inner root sheath (IRS) in apical region of hair follicles appeared to be hypertrophied. Cytoplasm of these cells was not stained by basic dyes and showed no birefringence. Cytoplasm of the IRS Henle cells was not stained by basic dyes either. These data indicate that keratinization of the IRS cells is disturbed in mutant homozygotes. The layer of outer root sheath in the we/we mice was thinner than in normal mice; this is probably due to hypertrophy of the IRS cells. The structure of differentiating cells of the hair shaft in normal and mutant mice was similar. The data obtained suggest that abnormal G1 hairs in we/we mice result from disturbance in IRS cells differentiation.     

Activation of Notch1 in the hair follicle leads to cell-fate switch and Mohawk alopecia

The Notch signaling pathway has been shown to control cell-fate decisions during mouse development. To study the role of Notch1 in epidermal differentiation and the development of the various cell types within the mouse hair follicle, we generated transgenic mice that express a constitutive activated form of Notch1 under the control of the involucrin promoter. Transgenic animals express the transgene in the suprabasal epidermal keratinocytes and inner root sheath of the hair follicle, and develop both skin and hair abnormalities. Notch1 overexpression leads to an increase of the differentiated cell compartment in the epidermis, delays inner root sheath differentiation, and leads to hair shaft abnormalities and alopecia associated with the anagen phase of the hair cycle.     

Hair follicular cell/organ culture in tissue engineering and regenerative medicine

Hair follicles are complex organs composed of the dermal papilla (DP), dermal sheath (DS), outer root sheath (ORS), inner root sheath (IRS) and hair shaft. Development of hair follicles begins towards the end of the first trimester of pregnancy and is controlled by epidermal–mesenchymal interaction (EMI), which is a signaling cascade between epidermal and mesenchymal cell populations. Hair grows in cycles of various phases. Specifically, anagen is the growth phase, catagen is the involuting or regressing phase and telogen is the resting or quiescent phase. Alopecia is not life threatening, but alopecia often causes severe mental stress. In addition, the number of individuals afflicted by alopecia patients has been increasing steadily. Currently there are two methods employed to treat alopecia, drug or natural substance therapy and human hair transplantation. Although drug or natural substance therapy may retard the progress of alopecia or prevent future hair loss, it may also accelerate hair loss when the medication is stopped after prolonged use. Conversely, the transplantation of human hair involves taking plugs of natural hair from areas in which occipital hair is growing and transplanting them to bald areas. However, the number of hairs that can be transplanted is limited in that only three such operations can generally be performed. To overcome such problems, many researchers have attempted to revive hair follicles by culturing hair follicle cells or mesenchymal cells in vitro and then implanting them in the treatment area.     

Inflammatory Mediator TAK1 Regulates Hair Follicle Morphogenesis and Anagen Induction Shown by Using Keratinocyte-Specific TAK1-Deficient Mice

Transforming growth factor-β-activated kinase 1 (TAK1) is a member of the NF-κB pathway and regulates inflammatory responses. We previously showed that TAK1 also regulates keratinocyte growth, differentiation, and apoptosis. However, it is unknown whether TAK1 has any role in epithelial–mesenchymal interactions. To examine this possibility, we studied the role of TAK1 in mouse hair follicle development and cycling as an instructive model system. By comparing keratinocyte-specific TAK1-deficient mice (Map3k7 ^fl/flK5-Cre) with control mice, we found that the number of hair germs (hair follicles precursors) in Map3k7 ^fl/flK5-Cre mice was significantly reduced at E15.5, and that subsequent hair follicle morphogenesis was retarded. Next, we analyzed the role of TAK1 in the cyclic remodeling in follicles by analyzing hair cycle progression in mice with a tamoxifen-inducible keratinocyte-specific TAK1 deficiency (Map3k7 ^fl/flK14-Cre-ER^T2). After active hair growth (anagen) was induced by depilation, TAK1 was deleted by topical tamoxifen application. This resulted in significantly retarded anagen development in TAK1-deficient mice. Deletion of TAK1 in hair follicles that were already in anagen induced premature, apoptosis-driven hair follicle regression, along with hair follicle damage. These studies provide the first evidence that the inflammatory mediator TAK1 regulates hair follicle induction and morphogenesis, and is required for anagen induction and anagen maintenance.     

A STUDY OF THE ULTRASTRUCTURAL LOCALIZATION OF HAIR KERATIN SYNTHESIS UTILIZING ELECTRON MICROSCOPIC AUTORADIOGRAPHY IN A MAGNETIC FIELD

The sites of the incorporation of labeled cystine into keratinizing structures were studied in electron microscopic autoradiographs. The tracer used was cystine labeled with S³⁵ emitting long-range ionizing particles. During exposure for 1 to 2 months, according to our method of electron microscopic autoradiography, emulsion-coated specimens were exposed to a static magnetic field which appeared to result in a marked increase in the number of reacted silver grains. In young Swiss mice receiving intraperitoneal injections at 1, 3, and 6 hours before biopsy, conventional autoradiography demonstrated that S³⁵-cystine was intensely localized in the keratogenous zone of anagen hair follicles, and that the radioactivity there increased in intensity progressively with time while the radioactivity in the hair bulb always remained very low. Our observations with electron microscopic autoradiography in a magnetic field appeared to indicate that at 3 and 6 hours after injection the S³⁵-cystine was directly and specifically incorporated into tonofibrils in the hair cortex and into amorphous keratin granules of the hair cuticle layer, possibly without any particular concentration of this substance in the other cellular components. There seemed to be an appreciable concentration of cystine in tonofibrils of the cuticle of the inner root sheath. However, trichohyalin granules in the hair medulla and inner root sheath failed to show any evidence of cystine concentration. The improved sensitivity of the electron microscopic autoradiography with S³⁵-cystine appeared to be partly due to the application of a static magnetic field. However, the reason for this could not be explained theoretically.     

WNT signaling in the control of hair growth and structure

Characterization of the molecular pathways controlling differentiation and proliferation in mammalian hair follicles is central to our understanding of the regulation of normal hair growth, the basis of hereditary hair loss diseases, and the origin of follicle-based tumors. We demonstrate that the proto-oncogene Wnt3, which encodes a secreted paracrine signaling molecule, is expressed in developing and mature hair follicles and that its overexpression in transgenic mouse skin causes a short-hair phenotype due to altered differentiation of hair shaft precursor cells, and cyclical balding resulting from hair shaft structural defects and associated with an abnormal profile of protein expression in the hair shaft. A putative effector molecule for WNT3 signaling, the cytoplasmic protein Dishevelled 2 (DVL2), is normally present at high levels in a subset of cells in the outer root sheath and in precursor cells of the hair shaft cortex and cuticle which lie immediately adjacent to Wnt3-expressing cells. Overexpression of Dvl2 in the outer root sheath mimics the short-hair phenotype produced by overexpression of Wnt3, supporting the hypothesis that Wnt3 and Dvl2 have the potential to act in the same pathway in the regulation of hair growth. These experiments demonstrate a previously unrecognized role for WNT signaling in the control of hair growth and structure, as well as presenting the first example of a mammalian phenotype resulting from overexpression of a Dvl gene and providing an accessible in vivo system for analysis of mammalian WNT signaling pathways.     

BMPR1A signaling is necessary for hair follicle cycling and hair shaft differentiation in mice

Interactions between ectodermal and mesenchymal extracellular signaling pathways regulate hair follicle (HF) morphogenesis and hair cycling. Bone morphogenetic proteins (BMPs) are known to be important in hair follicle development by affecting the local cell fate modulation. To study the role of BMP signaling in the HF, we disrupted Bmpr1a, which encodes the BMP receptor type IA (BMPR1A) in an HF cell-specific manner, using the Cre/loxP system. We found that the differentiation of inner root sheath, but not outer root sheath, was severely impaired in mutant mice. The number of HFs was reduced in the dermis and subcutaneous tissue, and cycling epithelial cells were reduced in mutant mice HFs. Our results strongly suggest that BMPR1A signaling is essential for inner root sheath differentiation and is indispensable for HF renewal in adult skin.     

Essential roles of BMPR-IA signaling in differentiation and growth of hair follicles and in skin tumorigenesis

Hair differentiation and growth are controlled by complex reciprocal signaling between epithelial and mesenchymal cells. To better understand the requirement and molecular mechanism of BMP signaling in hair follicle development, we performed genetic analyses of bone morphogenetic protein receptor 1A (BMPR-IA) function during hair follicle development by using a conditional knockout approach. The conditional mutation of Bmpr1a in ventral limb ectoderm and its derivatives (epidermis and hair follicles) resulted in a lack of hair outgrowth from the affected skin regions. Mutant hair follicles exhibited abnormal morphology and lacked hair formation and pigment deposition during anagen. The timing of the hair cycle and the proliferation of hair matrix cells were also affected in the mutant follicles. We demonstrate that signaling via epithelial BMPR-IA is required for differentiation of both hair shaft and inner root sheath from hair matrix precursor cells in anagen hair follicles but is dispensable for embryonic hair follicle induction. Surprisingly, aberrant de novo hair follicle morphogenesis together with hair matrix cell hyperplasia was observed in the absence of BMPR-IA signaling within the affected skin of adult mutants. They developed hair follicle tumors from 3 months of age, indicating that inactivation of epidermal BMPR-IA signaling can lead to hair tumor formation. Taken together, our data provide genetic evidence that BMPR-IA signaling plays critical and multiple roles in controlling cell fate decisions or maintenance, proliferation, and differentiation during hair morphogenesis and growth, and implicate Bmpr1a as a tumor suppressor in skin tumorigenesis.