Ruth Schmidt-Ullrich

The Hair Follicle as a Dynamic Miniorgan

Hair is a primary characteristic of mammals, and exerts a wide range of functions including thermoregulation, physical protection, sensory activity, and social interactions. The hair shaft consists of terminally differentiated keratinocytes that are produced by the hair follicle. Hair follicle development takes place during fetal skin development and relies on tightly regulated ectodermal-mesodermal interactions. After birth, mature and actively growing hair follicles eventually become anchored in the subcutis, and periodically regenerate by spontaneously undergoing repetitive cycles of growth (anagen), apoptosis-driven regression (catagen), and relative quiescence (telogen). Our molecular understanding of hair follicle biology relies heavily on mouse mutants with abnormalities in hair structure, growth, and/or pigmentation. These mice have allowed novel insights into important general molecular and cellular processes beyond skin and hair biology, ranging from organ induction, morphogenesis and regeneration, to pigment and stem cell biology, cell proliferation, migration and apoptosis. In this review, we present basic concepts of hair follicle biology and summarize important recent advances in the field.

Glucocorticoids Suppress Bone Formation by Attenuating Osteoblast Differentiation via the Monomeric Glucocorticoid Receptor

Development of osteoporosis severely complicates long-term glucocorticoid (GC) therapy. Using a Cre-transgenic mouse line, we now demonstrate that GCs are unable to repress bone formation in the absence of glucocorticoid receptor (GR) expression in osteoblasts as they become refractory to hormone-induced apoptosis, inhibition of proliferation, and differentiation. In contrast, GC treatment still reduces bone formation in mice carrying a mutation that only disrupts GR dimerization, resulting in bone loss in vivo, enhanced apoptosis, and suppressed differentiation in vitro. The inhibitory GC effects on osteoblasts can be explained by a mechanism involving suppression of cytokines, such as interleukin 11, via interaction of the monomeric GR with AP-1, but not NF-kappaB. Thus, GCs inhibit cytokines independent of GR dimerization and thereby attenuate osteoblast differentiation, which accounts, in part, for bone loss during GC therapy.

Reciprocal Requirements for EDA/EDAR/NF-κB and Wnt/β-Catenin Signaling Pathways in Hair Follicle Induction

Wnt/beta-catenin and NF-kappaB signaling mechanisms provide central controls in development and disease, but how these pathways intersect is unclear. Using hair follicle induction as a model system, we show that patterning of dermal Wnt/beta-catenin signaling requires epithelial beta-catenin activity. We find that Wnt/beta-catenin signaling is absolutely required for NF-kappaB activation, and that Edar is a direct Wnt target gene. Wnt/beta-catenin signaling is initially activated independently of EDA/EDAR/NF-kappaB activity in primary hair follicle primordia. However, Eda/Edar/NF-kappaB signaling is required to refine the pattern of Wnt/beta-catenin activity, and to maintain this activity at later stages of placode development. We show that maintenance of localized expression of Wnt10b and Wnt10a requires NF-kappaB signaling, providing a molecular explanation for the latter observation, and identify Wnt10b as a direct NF-kappaB target. These data reveal a complex interplay and interdependence of Wnt/beta-catenin and EDA/EDAR/NF-kappaB signaling pathways in initiation and maintenance of primary hair follicle placodes.

Requirement of Nuclear Factor-κB in Angiotensin II– and Isoproterenol-Induced Cardiac Hypertrophy In Vivo

Background—In vitro experiments have proposed a role of nuclear factor-&kgr;B (NF-&kgr;B), a transcription factor, in cardiomyocyte hypertrophy and protection against apoptosis. Currently, the net effect on cardiac remodeling in vivo under common stress stimuli is unclear. Methods and Results—We have generated mice with cardiomyocyte-restricted expression of the NF-&kgr;B super-repressor I&kgr;Bα&Dgr;N (&Dgr;NMHC) using the Cre/lox technique. &Dgr;NMHC mice displayed an attenuated hypertrophic response compared with control mice on infusion of angiotensin II (Ang II) or isoproterenol by micro-osmotic pumps, as determined by echocardiography (left ventricular wall dimensions: control plus Ang II, ×1.5±0.1 versus sham; &Dgr;NMHC plus Ang II, ×1.1±0.1 versus sham; P<0.05; n≥9), heart weight, and histological analysis. Real-time reverse-transcriptase polymerase chain reaction showed significantly reduced expression of hypertrophy markers β-myosin heavy chain and atrial natriuretic peptide in Ang II–treated &Dgr;NMHC mice (P<0.05 versus control plus Ang II; n=4). Neither cardiomyocyte apoptosis nor left ventricular dilatation was observed. In cultured adult rat cardiomyocytes, NF-&kgr;B DNA binding activity was increased by both Ang II– and interleukin-6–related cytokines. The latter are known to be released by cardiac fibroblasts on Ang II stimulation and thus could locally increase the NF-&kgr;B response of cardiomyocytes. Finally, results from in vitro and in vivo experiments suggest a role for NF-&kgr;B in the regulation of prohypertrophic interleukin-6 receptor gp130 on mRNA levels. Conclusions—These results indicate that targeted inhibition of NF-&kgr;B in cardiomyocytes in vivo is sufficient to impair Ang II– and isoproterenol-induced hypertrophy without increasing the susceptibility to apoptosis.

Activation of β-catenin signaling programs embryonic epidermis to hair follicle fate

β-Catenin signaling is required for hair follicle development, but it is unknown whether its activation is sufficient to globally program embryonic epidermis to hair follicle fate. To address this, we mutated endogenous epithelial β-catenin to a dominant-active form in vivo. Hair follicle placodes were expanded and induced prematurely in activated β-catenin mutant embryos, but failed to invaginate or form multilayered structures. Eventually, the entire epidermis adopted hair follicle fate, broadly expressing hair shaft keratins in place of epidermal stratification proteins. Mutant embryonic skin was precociously innervated, and displayed prenatal pigmentation, a phenomenon never observed in wild-type controls. Thus,β -catenin signaling programs the epidermis towards placode and hair shaft fate at the expense of epidermal differentiation, and activates signals directing pigmentation and innervation. In transcript profiling experiments, we identified elevated expression of Sp5, a direct β-catenin target and transcriptional repressor. We show that Sp5 normally localizes to hair follicle placodes and can suppress epidermal differentiation gene expression. We identified the pigmentation regulators Foxn1, Adamts20 and Kitl, and the neural guidance genes Sema4c, Sema3c, Unc5b and Unc5c, as potential mediators of the effects of β-catenin signaling on pigmentation and innervation. Our data provide evidence for a new paradigm in which, in addition to promoting hair follicle placode and hair shaft fate, β-catenin signaling actively suppresses epidermal differentiation and directs pigmentation and nerve fiber growth. Controlled downregulation of β-catenin signaling is required for normal placode patterning within embryonic ectoderm, hair follicle downgrowth, and adoption of the full range of follicular fates.

NF-κB transmits Eda A1/EdaR signalling to activate Shh and cyclin D1 expression, and controls post-initiation hair placode down growth

A novel function of NF-κB in the development of most ectodermal appendages, including two types of murine pelage hair follicles, was detected in a mouse model with suppressed NF-κB activity (cIκBαΔN). However, the developmental processes regulated by NF-κB in hair follicles has remained unknown. Furthermore, the similarity between the phenotypes of cIκBAΔN mice and mice deficient in Eda A1 (tabby) or its receptor EdaR (downless) raised the issue of whether in vivo NF-κB regulates or is regulated by these novel TNF family members. We now demonstrate that epidermal NF-κB activity is first observed in placodes of primary guard hair follicles at day E14.5, and that in vivo NF-κB signalling is activated downstream of Eda A1 and EdaR. Importantly, ectopic signals which activate NF-κB can also stimulate guard hair placode formation, suggesting a crucial role for NF-κB in placode development. In downless and cIκBαΔN mice, placodes start to develop, but rapidly abort in the absence of EdaR/NF-κB signalling. We show that NF-κB activation is essential for induction of Shh and cyclin D1 expression and subsequent placode down growth. However, cyclin D1 induction appears to be indirectly regulated by NF-κB, probably via Shh and Wnt. The strongly decreased number of hair follicles observed in cIκBαΔN mice compared with tabby mice, indicates that additional signals, such as TROY, must regulate NF-κB activity in specific hair follicle subtypes.

Vascular Endothelial Cell-Specific NF-κB Suppression Attenuates Hypertension-Induced Renal Damage

Nuclear factor kappa B (NF-&kgr;B) participates in hypertension-induced vascular and target-organ damage. We tested whether or not endothelial cell–specific NF-&kgr;B suppression would be ameliorative. We generated Cre/lox transgenic mice with endothelial cell–restricted NF-&kgr;B super-repressor I&kgr;B&agr;&Dgr;N (Tie-1-&Dgr;N mice) overexpression. We confirmed cell-specific I&kgr;B&agr;&Dgr;N expression and reduced NF-&kgr;B activity after TNF-&agr; stimulation in primary endothelial cell culture. To induce hypertension with target-organ damage, we fed mice a high-salt diet and N(omega)-nitro-l-arginine-methyl-ester (L-NAME) and infused angiotensin (Ang) II. This treatment caused a 40-mm Hg blood pressure increase in both Tie-1-&Dgr;N and control mice. In contrast to control mice, Tie-1-&Dgr;N mice developed a milder renal injury, reduced inflammation, and less albuminuria. RT-PCR showed significantly reduced expression of the NF-&kgr;B targets VCAM-1 and ICAM-1, compared with control mice. Thus, the data demonstrate a causal link between endothelial NF-&kgr;B activation and hypertension-induced renal damage. We conclude that in vivo NF-&kgr;B suppression in endothelial cells stops a signaling cascade leading to reduced hypertension-induced renal damage despite high blood pressure.

Ectodysplasin regulates hormone-independent mammary ductal morphogenesis via NF-κB

Ductal growth of the mammary gland occurs in two distinct stages. The first round of branching morphogenesis occurs during embryogenesis, and the second round commences at the onset of puberty. Currently, relatively little is known about the genetic networks that control the initial phases of ductal expansion, which, unlike pubertal development, proceeds independent of hormonal input in female mice. Here we identify NF-κB downstream of the TNF-like ligand ectodysplasin (Eda) as a unique regulator of embryonic and prepubertal ductal morphogenesis. Loss of Eda, or inhibition of NF-κB, led to smaller ductal trees with fewer branches. On the other hand, overexpression of Eda caused a dramatic NF-κB–dependent phenotype in both female and male mice characterized by precocious and highly increased ductal growth and branching that correlated with enhanced cell proliferation. We have identified several putative transcriptional target genes of Eda/NF-κB, including PTHrP, Wnt10a, and Wnt10b, as well as Egf family ligands amphiregulin and epigen. We developed a mammary bud culture system that allowed us to manipulate mammary development ex vivo and found that recombinant PTHrP, Wnt3A, and Egf family ligands stimulate embryonic branching morphogenesis, suggesting that these pathways may cooperatively mediate the effects of Eda.

Regulation of Vascular Smooth Muscle Cell Proliferation Role of NF-κB Revisited

The transcription factor NF-&kgr;B regulates cell cycle progression and proliferation in a number of cell types. An important unresolved issue is the potential role of NF-&kgr;B in the proliferation of vascular smooth muscle cells (VSMCs) as a basis for the development of vascular disease. To investigate the contribution of NF-&kgr;B to mitogen-induced proliferation of VSMCs, a knock-in mouse model expressing the NF-&kgr;B superrepressor I&kgr;B&agr;&Dgr;N (cI&kgr;B&agr;&Dgr;N) was used. Comparing wild-type and I&kgr;B&agr;&Dgr;N-expressing VSMCs, we found that proliferation rates did not differ after mitogenic stimulation by platelet-derived growth-factor-BB (PDGF-BB) or serum. In line with this, NF-&kgr;B activation was not observed in VSMCs derived from transgenic mice expressing an NF-&kgr;B–dependent lacZ reporter (c(Igk)3conalacZ). We further show, that classical mitogenic signaling pathways (namely mitogen-activated protein kinase [MAPK] and the phosphatidyl-inositol-3-OH-kinase [PI3K] pathways) control VSMC proliferation, but independently of NF-&kgr;B activation. In contrast to VSMCs, mouse embryonic fibroblasts (MEFs) derived from I&kgr;B&agr;&Dgr;N-expressing mice showed significantly impaired proliferation rates after mitogenic stimulation. This was reflected by strongly impaired cyclin D1 expression in serum-stimulated MEFs derived from (cI&kgr;B&agr;&Dgr;N) mice. These results implicate that essential pathogenetic functions of NF-&kgr;B in the development of atherosclerosis involve apoptotic and inflammatory signaling of VSMCs rather than proliferation. They further provide genetic evidence for a cell-type restricted requirement of NF-&kgr;B in the control of cellular proliferation.

Ectodysplasin and Wnt pathways are required for salivary gland branching morphogenesis

The developing submandibular salivary gland (SMG) is a well-studied model for tissue interactions and branching morphogenesis. Its development shares similar features with other ectodermal appendages such as hair and tooth. The ectodysplasin (Eda) pathway is essential for the formation and function of several ectodermal organs. Mutations in the signaling components of the Eda pathway lead to a human syndrome known as hypohidrotic ectodermal dysplasia (HED), which is characterized by missing and malformed teeth, sparse hair and reduced sweating. Individuals with HED suffer also from dry mouth because of reduced saliva flow. In order to understand the underlying mechanism, we analyzed salivary gland development in mouse models with altered Eda pathway activities. We have found that Eda regulates growth and branching of the SMG via transcription factor NF-κB in the epithelium, and that the hedgehog pathway is an important mediator of Eda/NF-κB. We also sought to determine whether a similar reciprocal interplay between the Eda and Wnt/β-catenin pathways, which are known to operate in other skin appendages, functions in developing SMG. Surprisingly and unlike in developing hair follicles and teeth, canonical Wnt signaling activity did not colocalize with Edar/NF-κB in salivary gland epithelium. Instead, we observed high mesenchymal Wnt activity and show that ablation of mesenchymal Wnt signaling either in vitro or in vivo compromised branching morphogenesis. We also provide evidence suggesting that the effects of mesenchymal Wnt/β-catenin signaling are mediated, at least in part, through regulation of Eda expression.