The up-regulation of 14-3-3 proteins in Smad4 deficient epidermis and hair follicles at catagen

Each postnatal hair follicle (HF) perpetually goes through three phases: anagen, catagen, and telogen. The molecular signals that orchestrate the follicular transition between phases are still largely unknown. Our previous study shows that the keratinocyte specific Smad4 knockout mice exhibit progressive alopecia due to the mutant HFs failure to undergo programmed regression. To investigate the detailed molecular events controlling this process, the protein profiles of Smad4 mutant and control epidermal and HF keratinocytes were compared using 2-D difference gel electrophoresis (2-D DIGE) proteomic analysis. Eighty-six differentially expressed protein spots were identified by MALDI-TOF/TOF MS or ESI-MS/MS as 72 proteins, of which 29 proteins were found to be changed during the anagen-catagen transition of HFs in Smad4 mutants compared with the controls. The differentially expressed proteins represent a wide spectrum of functional classes such as keratin, the cytoskeleton, cellular growth and differentiation, ion combination and transfer, protein enzymes. Notably, we found that the 14-3-3sigma protein together with the 14-3-3zeta and 14-3-3beta proteins were significantly down-regulated only in wild-type keratinocytes but not in Smad4 mutant keratinocytes during the catagen phase, suggesting that increased expression of 14-3-3 proteins might contribute to the blockade of catagen initiation in Smad4 deficient HFs.     

Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles

We previously demonstrated that keratin 15 expressing cells present in the bulge region of hair follicles exhibit properties of adult stem cells. We have now established and characterized an immortalized adult epithelial stem cell line derived from cells isolated from the human hair follicle bulge region. Telogen hair follicles from human skin were microdissected to obtain an enriched population of keratin 15 positive skin stem cells. By expressing human papillomavirus 16 E6/E7 genes in these stem cells, we have been able to culture the cells for >30 passages and maintain a stable phenotype after 12 mo of continuous passage. The cell line was compared to primary stem cells for expression of stem cell specific proteins, for in vitro stem cell properties, and for their capacity to differentiate into different cell lineages. This new cell line, named Tel-E6E7 showed similar expression patterns to normal skin stem cells and maintained in vitro properties of stem cells. The cells can differentiate into epidermal, sebaceous gland, and hair follicle lineages. Intact beta-catenin dependent signaling, which is known to control in vivo hair differentiation in rodents, is maintained in this cell line. The Tel-E6E7 cell line may provide the basis for valid, reproducible in vitro models for studies on stem cell lineage determination and differentiation.     

Comparative substrate specificity analysis of recombinant human cathepsin V and cathepsin L

Cathepsins V and L have high identity and few structural differences. In this paper, we reported a comparative study of the hydrolytic activities of recombinant human cathepsins V and L using fluorescence resonance energy transfer peptides derived from Abz-KLRSSKQ-EDDnp (Abz = ortho-aminobenzoic acid and EDDnp = N-(2,4-dinitrophenyl)ethylenediamine). Five series of peptides were synthesized to map the S3 to S2' subsites. The cathepsin V subsites S1 and S3 present a broad specificity while cathepsin L has preference for positively charged residues. The S2 subsites of both enzymes require hydrophobic residues with preference for Phe and Leu. The S1' and S2' subsites of cathepsins V and L are less specific. Based on these data we designed substrates to explore the electrostatic potential differences of them. Finally, the kininogenase activities of these cathepsins were compared using synthetic human kininogen fragments. Cathepsin V preferentially released Lys-bradykinin while cathepsin L released bradykinin. This kininogenase activity by cathepsins V and L was also observed from human high and low molecular weight kininogens.     

Human cathepsin V protease participates in production of enkephalin and NPY neuropeptide neurotransmitters

Proteases are required for processing precursors into active neuropeptides that function as neurotransmitters for cell-cell communication. This study demonstrates the novel function of human cathepsin V protease for producing the neuropeptides enkephalin and neuropeptide Y (NPY). Cathepsin V is a human-specific cysteine protease gene. Findings here show that expression of cathepsin V in neuroendocrine PC12 cells and human neuronal SK-N-MC cells results in production of (Met)enkephalin from proenkephalin. Gene silencing of cathepsin V by siRNA in human SK-N-MC cells results in reduction of (Met)enkephalin by more than 80%, illustrating the prominent role of cathepsin V for neuropeptide production. In vitro processing of proenkephalin by cathepsin V occurs at dibasic residue sites to generate enkephalin-containing peptides and an ～24-kDa intermediate present in human brain. Cathepsin V is present in human brain cortex and hippocampus where enkephalin and NPY are produced and is present in purified human neuropeptide secretory vesicles. Colocalization of cathepsin V with enkephalin and NPY in secretory vesicles of human neuroblastoma cells was illustrated by confocal microscopy. Furthermore, expression of cathepsin V with proNPY results in NPY production. These findings indicate the unique function of human cathepsin V for producing enkephalin and NPY neuropeptides required for neurotransmission in health and neurological diseases.     

Actin bundles in human hair follicles as revealed by confocal laser microscopy

A confocal laser microscope was used to examine the distribution pattern of actin bundles in whole-mounts of human hair follicles stained with fluorescently labeled phalloidin. Actin bundles were found exclusively in the epithelial outer root sheath of the lower and middle portions of the follicle. In the growth stage, the lower follicle was characterized by well-developed actin bundles arranged circumferentially in the innermost and outermost cell layers of the outer root sheath. Actin bundles in the innermost cells were aligned end-to-end so that they formed complete circular bands surrounding the inner root sheath. In the outermost cells, actin bundles ran underneath the basal plasma membrane to which they attached at both ends. In contrast, in the quiescent stage, actin bundles in the lower follicle were disposed radially toward the follicle surface where they terminated perpendicular to the basal plasma membrane. In the middle follicle, circumferential actin bundles were found only in the intermediate layer of the outer root sheath throughout the hair cycle. Immunofluorescent anti-myosin and anti-α-actinin staining showed a striated pattern along actin bundles. Vinculin was localized at both ends of actin bundles, corresponding to the cell-to-cell or cell-to-substrate adherens junctions. Glycerinated follicles changed in shape on the addition of MgATP, suggesting a contraction of actin bundles. From these observations, we conclude that actin bundles in the hair follicle are comparable to stress fibers and that they serve as a tensile scaffold for the growth and integrity of the follicle. Received: 6 May 1995 / Accepted: 25 October 1995     

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.     

An activity-based probe for the determination of cysteine cathepsin protease activities in whole cells

We describe a novel diazomethylketone-containing irreversible inhibitor (BIL-DMK) which is specific for a subset of pharmaceutically important cysteine cathepsin proteases. BIL-DMK rapidly inactivates cathepsins B, F, K, L, S, and V in isolated enzyme assays and labels cathepsins in whole cells. The presence of catalytically active cathepsins B, L, and K or S was demonstrated using radioiodinated BIL-DMK in HepG2 (hepatoma), HIG82 (rabbit synoviocyte), and Ramos (B lymphoma) cell lines, respectively. The identity of each protein labeled was confirmed from the isoelectric point and molecular mass of the radioactive spots on two-dimensional gel and by comigration with each cathepsin as identified by immunoblotting. These cell lines were used to establish whole-cell enzyme occupancy assays to determine the potency of both irreversible and reversible inhibitors against each cathepsin in their native cellular lysosomal or endosomal environment. These whole-cell enzyme occupancy assays are useful to determine the cellular permeability of competing inhibitors and have the advantage of not requiring specific substrates for each cathepsin of interest.     

Internalization of exogenous cystatin F supresses cysteine proteases and induces the accumulation of single-chain cathepsin L by multiple mechanisms

Cystatin F is an unusual member of the cystatin family of protease inhibitors, which is made as an inactive dimer and becomes activated by proteolysis in the endo/lysosome pathway of the immune cells that produce it. However a proportion is secreted and can be taken up and activated by other cells. We show here that cystatin F acquired in this way induces a dramatic accumulation of the single-chain form of cathepsin L (CatL). Cystatin F was observed in the same cellular compartments as CatL and was tightly complexed with CatL as determined by co-precipitation studies. The observed accumulation of single-chain CatL was partly due to cystatin F-mediated inhibition of the putative single-chain to two-chain CatL convertase AEP/legumain and partly to general suppression of cathepsin activity. Thus, cystatin F stabilizes CatL leading to the dramatic accumulation of an inactive complex composed either of the single-chain or two-chain form depending on the capacity of cystatin F to inhibit AEP. Cross-transfer of cystatin F from one cell to another may therefore attenuate potentially harmful effects of excessive CatL activity while paradoxically, inducing accumulation of CatL protein. Finally, we confirmed earlier data (Beers, C., Honey, K., Fink, S., Forbush, K., and Rudensky, A. (2003) J. Exp. Med. 197, 169-179) showing a loss of CatL activity, but not of CatL protein, in macrophages activated with IFNγ. However, we found equivalent loss of CatL activity in wild type and cystatin F-null macrophages suggesting that an inhibitory activity other than cystatin F quenches CatL activity in activated macrophages.     

Expression of human cathepsin L or human cathepsin V in mouse thymus mediates positive selection of T helper cells in cathepsin L knock-out mice

A genetic deficiency of the cysteine protease cathepsin L (Ctsl) in mice results in impaired positive selection of conventional CD4+ T helper cells as a result of an incomplete processing of the MHC class II associated invariant chain or incomplete proteolytic generation of positively selecting peptide ligands. The human genome encodes, in contrast to the mouse genome, for two cathepsin L proteases, namely cathepsin L (CTSL) and cathepsin V (CTSV; alternatively cathepsin L2). In the human thymic cortex, CTSV is the predominately expressed protease as compared to CTSL or other cysteine cathepsins. In order to analyze the functions of CTSL and CTSV in the positive selection of CD4+ T cells we employed Ctsl knock-out mice crossed either with transgenic mice expressing CTSL under the control of its genuine human promoter or with transgenic mice expressing CTSV under the control of the keratin 14 (K14) promoter, which drives expression to the cortical epithelium. Both human proteases are expressed in the thymus of the transgenic mice, and independent expression of both CTSL and CTSV rescues the reduced frequency of CD4+ T cells in Ctsl-deficient mice. Moreover, the expression of the human cathepsins does not change the number of CD4+CD25+Foxp3+ regulatory T cells, but the normalization of the frequency of conventional CD4+ T cell in the transgenic mice results in a rebalancing of conventional T cells and regulatory T cells. We conclude that the functional differences of CTSL and CTSV in vivo are not mainly determined by their inherent biochemical properties, but rather by their tissue specific expression pattern.     

Daunomycin accumulation and induction of programmed cell death in rat hair follicles

The anthracycline antibiotic daunomycin (DM) is useful for the treatment of leukemia but has side-effects such as alopecia. Using immunocytochemistry, we show that, after a single i.v. injection, DM accumulates in the nuclei of matrix cells and in the outer root sheath of hair follicles. DM-positive matrix cells are detectable up to 48 h after injection and exhibit a characteristic granular morphology, which is not observed in saline-injected controls. TUNEL-staining has revealed that DM injection induces programmed cell death (PCD) in rat hair follicles. Cells undergoing PCD are detectable as late as 5 days postinjection in both the matrix and outer root sheath. Newly developed double-staining has shown that some of the DM-positive matrix cell nuclei are also TUNEL-positive. Staining for activated caspase-3 has demonstrated immunopositive cells following DM administration both in the matrix and in the outer root sheath. Ultrastructural immunocytochemistry has shown the presence of DM-positive cells with two different types of morphology. About half of the immunopositive cells exhibit a morphology typical of classical apoptosis (PCD type 1), whereas the other half show signs of autophagic cell death (PCD type 2). Interestingly, little, if any, DM accumulation or apoptosis has been detected in the dermal hair papillae. This may have a bearing on potential regeneration of the hair follicles. Thus, DM accumulates in a characteristic pattern in hair follicles. This accumulation is associated with the induction of two morphologically distinct forms of PCD.     

Merkel cell distribution in human hair follicles of the fetal and adult scalp

The distribution of Merkel cells in fetal and adult terminal hair follicles of human scalp was studied immunohistochemically using cytokeratin (CK) 20 as a specific Merkel cell marker. In hair follicles of adult scalp, abundant Merkel cells were found enriched in two belt-like clusters, one in the deep infundibulum and one in the isthmus region. No Merkel cells were found in the deep follicular portions including the bulb, or in the dermis. In early fetal hair follicles (bulbous peg stage), Merkel cells were only detected in the basal layer of the developing infundibulum but not in deeper follicular areas. In later stages, Merkel cells were also present in the isthmus and bulge. No Merkel cells were seen in the dermis around developing hair follicles. Nerve growth factor receptor was not only present in nerves but was found to be widely distributed within fetal skin. In adult skin, this receptor was localized to the basal cell layers of the outer root sheath of the bulb and the suprabulbar area, but was not detectable in the areas containing Merkel cells. The present study localizing Merkel cells within the permanent hair follicle structures close to their possible stem cells suggests that they have paracrine functions.     

The role of activins and follistatins in skin and hair follicle development and function

Investigations of the signalling between epithelial and mesenchymal compartments of skin during hair follicle initiation in utero and hair cycling have revealed the importance of the TGFβ superfamily in ectodermal organogenesis and morphogenesis. In particular the activins, their receptors and binding proteins such as follistatin, have been shown to be important regulators of cell proliferation, differentiation and apoptosis in hair follicle initiation, hair cycling, normal skin homeostasis and wound healing. Transgenic mice lacking various components of the activin signalling pathways display varying ectodermal pathologies including altered pelage hair follicle initiation. This review summarises the activin signal transduction pathways and the interactions between activins and other TGFβ signalling systems during hair follicle formation, hair growth cycling, skin function and wound healing.     

Detection of Bim and Puma in mouse hair follicles using immunofluorescence and TUNEL assay double staining

Apoptosis in hair follicles often is studied under pathological conditions; little is known about apoptotic mechanisms during normal hair follicle formation and maintenance. We investigated proteins of intrinsic apoptotic pathway, Bim and Puma, during hair follicle development and the first catagen stage using immunofluorescence to describe their expression patterns and to correlate them with apoptosis as determined by TUNEL assay. Both proteins were found in developing follicles. Bim and Puma overlapped apoptosis only partially during physiological apoptotic stage and they were present in non-apoptotic parts of the follicles. Our findings suggest that these primary apoptotic molecules participate in postnatal development and maintenance of hair follicles.     

Two optical coherence tomography systems detect topical gold nanoshells in hair follicles,sweat ducts and measure epidermis

Optical coherence tomography (OCT) is an established imaging technology for in vivo skin investigation. Topical application of gold nanoshells (GNS) provides contrast enhancement in OCT by generating a strong hyperreflective signal from hair follicles and sweat glands, which are the natural skin openings. This study explores the utility of 150 nm diameter GNS as contrast agent for OCT imaging. GNS was massaged into skin and examined in four skin areas of 11 healthy volunteers. A commercial OCT system and a prototype with 3 μm resolution (UHR‐OCT) were employed to detect potential benefits of increased resolution and variability in intensity generated by the GNS. In both OCT‐systems GNS enhanced contrast from hair follicles and sweat ducts. Highest average penetration depth of GNS was in armpit 0.64 mm ± SD 0.17, maximum penetration depth was 1.20 mm in hair follicles and 15 to 40 μm in sweat ducts. Pixel intensity generated from GNS in hair follicles was significantly higher in UHR‐OCT images (P = .002) and epidermal thickness significantly lower 0.14 vs 0.16 mm (P = .027). This study suggests that GNSs are interesting candidates for increasing sensitivity in OCT diagnosis of hair and sweat gland disorders and demonstrates that choice of OCT systems influences results.      

Optimization of dipeptidic inhibitors of cathepsin L for improved Toxoplasma gondii selectivity and CNS permeability

The neurotropic protozoan Toxoplasma gondii is the second leading cause of death due to foodborne illness in the US, and has been designated as one of five neglected parasitic infections by the Center for Disease Control and Prevention. Currently, no treatment options exist for the chronic dormant-phase Toxoplasma infection in the central nervous system (CNS). T. gondii cathepsin L (TgCPL) has recently been implicated as a novel viable target for the treatment of chronic toxoplasmosis. In this study, we report the first body of SAR work aimed at developing potent inhibitors of TgCPL with selectivity vs the human cathepsin L. Starting from a known inhibitor of human cathepsin L, and guided by structure-based design, we were able to modulate the selectivity for Toxoplasma vs human CPL by nearly 50-fold while modifying physiochemical properties to be more favorable for metabolic stability and CNS penetrance. The overall potency of our inhibitors towards TgCPL was improved from 2?μM to as low as 110?nM and we successfully demonstrated that an optimized analog 18b is capable of crossing the BBB (0.5?brain/plasma). This work is an important first step toward development of a CNS-penetrant probe to validate TgCPL as a feasible target for the treatment of chronic toxoplasmosis.     

Tmprss3, a transmembrane serine protease deficient in human DFNB8/10 deafness, is critical for cochlear hair cell survival at the onset of hearing

Mutations in the type II transmembrane serine protease 3 (TMPRSS3) gene cause non-syndromic autosomal recessive deafness (DFNB8/10), characterized by congenital or childhood onset bilateral profound hearing loss. In order to explore the physiopathology of TMPRSS3 related deafness, we have generated an ethyl-nitrosourea-induced mutant mouse carrying a protein-truncating nonsense mutation in Tmprss3 (Y260X) and characterized the functional and histological consequences of Tmprss3 deficiency. Auditory brainstem response revealed that wild type and heterozygous mice have normal hearing thresholds up to 5 months of age, whereas Tmprss3(Y260X) homozygous mutant mice exhibit severe deafness. Histological examination showed degeneration of the organ of Corti in adult mutant mice. Cochlear hair cell degeneration starts at the onset of hearing, postnatal day 12, in the basal turn and progresses very rapidly toward the apex, reaching completion within 2 days. Given that auditory and vestibular deficits often co-exist, we evaluated the balancing abilities of Tmprss3(Y260X) mice by using rotating rod and vestibular behavioral tests. Tmprss3(Y260X) mice effectively displayed mild vestibular syndrome that correlated histologically with a slow degeneration of saccular hair cells. In situ hybridization in the developing inner ear showed that Tmprss3 mRNA is localized in sensory hair cells in the cochlea and the vestibule. Our results show that Tmprss3 acts as a permissive factor for cochlear hair cells survival and activation at the onset of hearing and is required for saccular hair cell survival. This mouse model will certainly help to decipher the molecular mechanisms underlying DFNB8/10 deafness and cochlear function.     

Role of Sonic hedgehog signaling in epithelial and mesenchymal development of hair follicles in an organ culture of embryonic mouse skin

Studies with gene knockout mice have shown that Sonic hedgehog (Shh) is required for early development of hair follicles, but the role of this gene in the late stages of follicle development is not clear. By using an organ culture system of embryonic mouse skin, the role of Shh signaling in the early and late stages of follicle development was investigated. In the early stage of follicle development, the downward growth of the follicular epithelium was suppressed by cyclopamine, an inhibitor of Shh signaling, and accelerated by recombinant Shh. In addition, cyclopamine impaired dermal papilla formation, accompanied by the rearrangement of papilla cells, but not the elongation of the follicular epithelium at the later stage. These results suggest that Shh signaling is required for the proliferation of epithelial cells in the early development of hair follicles and for the morphogenetic movement of mesenchymal cells at the later stage of follicle development.     

The targeted overexpression of a Claudin mutant in the epidermis of transgenic mice elicits striking epidermal and hair follicle abnormalities

Skin is one of the largest organs of the body, and is formed during development through a highly orchestrated process involving mesenchymal-epithelial interactions, cell commitment, and terminal differentiation. It protects against microorganism invasion and UV irradiation, inhibits water loss, regulates body temperature, and is an important part of the immune system. Using transgenic mouse technology, we have demonstrated that Claudin (Cldn)-containing tight junctions (TJs) are intricately involved in cell signaling during epidermal differentiation and that an epidermal suprabasal overexpression of Cldn6 results in a perturbed epidermal terminal differentiation program with distinct phenotypic abnormalities. To delineate the role of the Cldn cytoplasmic tail domain in epidermal differentiation, we engineered transgenic mice targeting the overexpression of a Cldn6 cytoplasmic tail-truncation mutant in the epidermis. Transgenic mice were characterized by a lethal barrier dysfunction in addition to the existence of hyperproliferative squamous invaginations/cysts replacing hair follicles. Immunohistochemical analysis revealed an epidermal cytoplasmic accumulation of Cldn6, Cldn11, Cldn12, and Cldn18, downregulation of Cldn1 and aberrant expression of various classical markers of epidermal differentiation; namely the basal keratins as well as K1, involucrin, loricrin, and filaggrin. Collectively these studies suggest an important role for Cldns in epidermal/hair follicle differentiation programs likely involving cross talk to signaling pathways (e.g., Notch) directing cell fate selection and differentiation.