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Dental enamel formation is coordinated by ameloblast differentiation, production of enamel matrix proteins, and crystal growth. The factors regulating ameloblast differentiation are not fully understood. Here we show that the high mobility group N (HMGN) nucleosomal binding proteins modulate the rate of ameloblast differentiation and enamel formation. We found that HMGN1 and HMGN2 proteins are downregulated during mouse ameloblast differentiation. Genetically altered mice lacking HMGN1 and HMGN2 proteins show faster ameloblast differentiation and a higher rate of enamel deposition in mice molars and incisors. In vitro differentiation of induced pluripotent stem cells to dental epithelium cells showed that HMGN proteins modulate the expression and chromatin accessibility of ameloblast-specific genes and affect the binding of transcription factors epiprofin and PITX2 to ameloblast-specific genes. Our results suggest that HMGN proteins regulate ameloblast differentiation and enamel mineralization by modulating lineage-specific chromatin accessibility and transcription factor binding to ameloblast regulatory sites. [ABSTRACT FROM AUTHOR]

The magnitude and variation of tooth pair compliance affects tooth loading and gear dynamics significantly. This paper presents an improved fillet/foundation compliance analysis based on the theory of Muskhelishvili applied to circular elastic rings. Assuming linear and constant stress variations at root circle, the above theory makes it possible to derive an analytical formula for gear body-induced tooth deflections which can be directly integrated into gear computer codes. The corresponding results are in very good agreement with those from finite element models and the formula is proved to be superior to Weber's widely used equation, especially for large gears. [DOI: 10.1115/1.1758252]

Introduction: Interferon-gamma (IFN-γ) is pivotal in orchestrating immune responses during healthy pregnancy. However, its dysregulation, often due to autoimmunity, infections, or chronic inflammatory conditions, is implicated in adverse reproductive outcomes such as pregnancy failure or infertility. Additionally, the underlying immunological mechanisms remain elusive., Methods: Here, we explore the impact of systemic IFN-γ elevation on cytotoxic T cell responses in female reproduction utilizing a systemic lupus-prone mouse model with impaired IFN-γ degradation., Results: Our findings reveal that heightened IFN-γ levels triggered the infiltration of CD8 + T cells in the pituitary gland and female reproductive tract (FRT), resulting in prolactin deficiency and subsequent infertility. Furthermore, we demonstrate that chronic IFN-γ elevation increases effector memory CD8 + T cells in the murine ovary and uterus., Discussion: These insights broaden our understanding of the role of elevated IFN-γ in female reproductive dysfunction and suggest CD8 + T cells as potential immunotherapeutic targets in female reproductive disorders associated with chronic systemic IFN-γ elevation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Bafor, Erwin-Cohen, Martin, Baker, Kimmel, Duverger, Fenimore, Ramba, Spindel, Hess, Sanford, Lazarevic, Benayoun, Young and Valencia.)

Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare autosomal recessive disorder caused by mutations in FGF23 , GALNT3 , KLOTHO , or FGF23 autoantibodies. Prominent features include high blood phosphate and calcific masses, usually adjacent to large joints. Dental defects have been reported, but not systematically described. Seventeen patients with HFTC followed at the National Institutes of Health underwent detailed clinical, biochemical, molecular, and dental analyses. Studies of teeth included intraoral photos and radiographs, high-resolution μCT, histology, and scanning electron microscopy (SEM). A scoring system was developed to assess the severity of tooth phenotype. Pulp calcification was found in 13 of 14 evaluable patients. Short roots and midroot bulges with apical thinning were present in 12 of 13 patients. Premolars were most severely affected. μCT analyses of five HFTC teeth revealed that pulp density increased sevenfold, whereas the pulp volume decreased sevenfold in permanent HFTC teeth compared with age- and tooth-matched control teeth. Histology revealed loss of the polarized odontoblast cell layer and an obliterated pulp cavity that was filled with calcified material. The SEM showed altered pulp and cementum structures, without differences in enamel or dentin structures, when compared with control teeth. This study defines the spectrum and confirms the high penetrance of dental features in HFTC. The phenotypes appear to be independent of genetic/molecular etiology, suggesting hyperphosphatemia or FGF23 deficiency may be the pathomechanistic driver, with prominent effects on root and pulp structures, consistent with a role of phosphate and/or FGF23 in tooth development. Given the early appearance and high penetrance, cognizance of HFTC-related features may allow for earlier diagnosis and treatment. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research., Competing Interests: The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article., (© 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.)

Background Loeys-Dietz syndrome (LDS), an autosomal dominant rare connective tissue disorder, has multisystemic manifestations, characterised by vascular tortuosity, aneurysms and craniofacial manifestations. Based on the associated gene mutations along the transforming growth factor-beta (TGF-β) pathway, LDS is presently classified into six subtypes. Methods We present the oro-dental features of a cohort of 40 patients with LDS from five subtypes. Results The most common oro-dental manifestations were the presence of a high-arched and narrow palate, and enamel defects. Other common characteristics included bifid uvula, submucous cleft palate, malocclusion, dental crowding and delayed eruption of permanent teeth. Both deciduous and permanent teeth had enamel defects in some individuals. We established a grading system to measure the severity of enamel defects, and we determined that the severity of the enamel anomalies in LDS is subtype-dependent. In specific, patients with TGF-β receptor II mutations (LDS2) presented with the most severe enamel defects, followed by patients with TGF-β receptor I mutations (LDS1). LDS2 patients had higher frequency of oro-dental deformities in general. Across all five subtypes, as well as within each subtype, enamel defects exhibited incomplete penetrance and variable expression, which is not associated with the location of the gene mutations. Conclusion This study describes, in detail, the oro-dental manifestations in a cohort of LDS, and we conclude that LDS2 has the most severely affected phenotype. This extensive characterisation, as well as some identified distinguishing features can significantly aid dental and medical care providers in the diagnosis and clinical management of patients with this rare connective tissue disorder., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Loeys-Dietz syndrome (LDS) is a rare connective tissue disorder whose oral manifestations and dental phenotypes have not been well-characterized. The aim of this study was to explore the influence of oral manifestations on oral health-related quality of life (OHRQoL) in LDS patients., Material and Methods: LDS subjects were assessed by the craniofacial team at the National Institutes of Health Clinical Center Dental Clinic between June 2015 and January 2018. Oral Health Impact Profile (OHIP-14) questionnaire, oral health self-care behavior questionnaire and a comprehensive dental examination were completed for each subject. OHRQoL was assessed using the OHIP-14 questionnaire with higher scores corresponding to worse OHRQoL. Regression models were used to determine the relationship between each oral manifestation and the OHIP-14 scores using a level of significance of p ≤ 0.05., Results: A total of 33 LDS subjects (51.5% female) aged 3-57 years (19.6 ± 15.1 years) were included in the study. The OHIP-14 scores (n = 33) were significantly higher in LDS subjects (6.30 [SD 6.37]) when compared to unaffected family member subjects (1.50 [SD 2.28], p < 0.01), and higher than the previously reported scores of the general U.S. population (2.81 [SD 0.12]). Regarding oral health self-care behavior (n = 32), the majority of LDS subjects reported receiving regular dental care (81%) and maintaining good-to-excellent daily oral hygiene (75%). Using a crude regression model, worse OHRQoL was found to be associated with dental hypersensitivity (β = 5.24; p < 0.05), temporomandibular joints (TMJ) abnormalities (β = 5.92; p < 0.01), self-reported poor-to-fair oral health status (β = 6.77; p < 0.01), and cumulation of four or more oral manifestations (β = 7.23; p < 0.001). Finally, using a parsimonious model, self-reported poor-to-fair oral health status (β = 5.87; p < 0.01) and TMJ abnormalities (β = 4.95; p < 0.01) remained significant., Conclusions: The dental hypersensitivity, TMJ abnormalities, self-reported poor-to-fair oral health status and cumulation of four-or-more oral manifestations had significant influence on worse OHRQoL. Specific dental treatment guidelines are necessary to ensure optimal quality of life in patients diagnosed with LDS.

A highly specialized cytoskeletal protein, keratin 75 (K75), expressed primarily in hair follicles, nail beds, and lingual papillae, was recently discovered in dental enamel, the most highly mineralized hard tissue in the human body. Among many questions this discovery poses, the fundamental question regarding the trafficking and secretion of this protein, which lacks a signal peptide, is of an utmost importance. Here, we present evidence that K75 is expressed during the secretory stage of enamel formation and is present in the forming enamel matrix. We further show that K75 is secreted together with major enamel matrix proteins amelogenin and ameloblastin, and it was detected in Golgi and the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) but not in rough ER (rER). Inhibition of ER-Golgi transport by brefeldin A did not affect the association of K75 with Golgi, whereas ameloblastin accumulated in rER, and its transport from rER into Golgi was disrupted. Together, these results indicate that K75, a cytosolic protein lacking a signal sequence, is secreted into the forming enamel matrix utilizing portions of the conventional ER-Golgi secretory pathway. To the best of our knowledge, this is the first study providing insights into mechanisms of keratin secretion.

DLX3 is essential for tooth enamel development and is so far the only transcription factor known to be mutated in a syndromic form of amelogenesis imperfecta. Through conditional deletion of Dlx3 in the dental epithelium in mouse, we have previously established the involvement of DLX3 in enamel pH regulation, as well as in controlling the expression of sets of keratins that contribute to enamel rod sheath formation. Here, we show that the decussation pattern of enamel rods was lost in conditional knockout animals, suggesting that DLX3 controls the coordinated migration of ameloblasts during enamel secretion. We further demonstrate that DLX3 regulates the expression of some components of myosin II complexes potentially involved in driving the movement of ameloblasts that leads to enamel rod decussation.

Pachyonychia congenita (PC) is a cutaneous disorder primarily characterized by nail dystrophy and painful palmoplantar keratoderma. PC is caused by mutations in KRT6A, KRT6B, KRT6C, KRT16, and KRT17, a set of keratin genes expressed in the nail bed, palmoplantar epidermis, oral mucosal epithelium, hair follicle and sweat gland. RNA-seq analysis revealed that all PC-associated keratins (except for Krt6c that does exist in the mouse genome) are expressed in the mouse enamel organ. We further demonstrated that these keratins are produced by ameloblasts and are incorporated into mature human enamel. Using genetic and intraoral examination data from 573 adults and 449 children, we identified several missense polymorphisms in KRT6A, KRT6B and KRT6C that lead to a higher risk for dental caries. Structural analysis of teeth from a PC patient carrying a p.Asn171Lys substitution in keratin-6a (K6a) revealed disruption of enamel rod sheaths resulting in altered rod shape and distribution. Finally, this PC-associated substitution as well as more frequent caries-associated SNPs, found in two of the KRT6 genes, that result in p.Ser143Asn substitution (rs28538343 in KRT6B and rs151117600 in KRT6C), alter the assembly of K6 filaments in ameloblast-like cells. These results identify a new set of keratins involved in tooth enamel formation, distinguish novel susceptibility loci for tooth decay and reveal additional clinical features of pachyonychia congenita.

Patients with tricho-dento-osseous (TDO) syndrome, an ectodermal dysplasia caused by mutations in the homeodomain transcription factor DLX3, exhibit enamel hypoplasia and hypomineralization. Here we used a conditional knockout mouse model to investigate the developmental and molecular consequences of Dlx3 deletion in the dental epithelium in vivo. Dlx3 deletion in the dental epithelium resulted in the formation of chalky hypomineralized enamel in all teeth. Interestingly, transcriptomic analysis revealed that major enamel matrix proteins and proteases known to be involved in enamel secretion and maturation were not affected significantly by Dlx3 deletion in the enamel organ. In contrast, expression of several ion transporters and carbonic anhydrases known to play an important role in enamel pH regulation during maturation was significantly affected in enamel organs lacking DLX3. Most of these affected genes showed binding of DLX3 to their proximal promoter as evidenced by chromatin immunoprecipitation sequencing (ChIP-seq) analysis on rat enamel organ. These molecular findings were consistent with altered pH staining evidenced by disruption of characteristic pH oscillations in the enamel. Taken together, these results show that DLX3 is indispensable for the regulation of ion transporters and carbonic anhydrases during the maturation stage of amelogenesis, exerting a crucial regulatory function on pH oscillations during enamel mineralization. © 2016 American Society for Bone and Mineral Research., (© 2016 American Society for Bone and Mineral Research.)

Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis of many of these disorders remains unknown. In the present study, we aimed at elucidating the genetic basis of a new form of ED featuring facial dysmorphism, scalp hypotrichosis and hypodontia. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR segregating with the disease phenotype in 3 families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in murine hair and tooth development. Pathway analysis confirmed that down-regulation of TSPEAR in keratinocytes is likely to affect Notch signaling. Accordingly, using a luciferase-based reporter assay, we showed that TSPEAR knock-down is associated with decreased Notch signaling. In addition, NOTCH1 protein expression was reduced in patient scalp skin. Moreover, TSPEAR silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch signaling pathway., Competing Interests: The authors have declared that no competing interests exist.

Dental enamel is the hardest tissue in the human body, and although it starts as a tissue rich in proteins, by the time of eruption of the tooth in the oral cavity only a small fraction of the protein remains. While this organic matrix of enamel represents less than 1% by weight it plays essential roles in improving both toughness and resilience to chemical attacks. Despite the fact that the first studies of the enamel matrix began in the 19th century, its exact composition and mechanisms of its function remain poorly understood. It was proposed that keratin or a keratin-like primitive epithelial component exists in mature enamel, however due to the extreme insolubility of its organic matrix the presence of keratins there was never clearly established. We have recently identified expression of a number of hair keratins in ameloblasts, the enamel secreting cells, and demonstrated their incorporation into mature enamel. Mutation in epithelial hair keratin KRT75 leads to a skin condition called pseudofollicularis barbae. Carriers of this mutation have an altered enamel structure and mechanical properties. Importantly, these individuals have a much higher prevalence of caries. To the best of our knowledge, this is the first study showing a direct link between a mutation in a protein-coding region of a gene and increased caries rates. In this paper we present an overview of the evidence of keratin-like material in enamel that has accumulated over the last 150years. Furthermore, we propose potential mechanisms of action of KTR75 in enamel and highlight the clinical implications of the link between mutations in KRT75 and caries. Finally, we discuss the potential use of keratins for enamel repair., (Published by Elsevier B.V.)

Tooth enamel is the hardest substance in the human body and has a unique combination of hardness and fracture toughness that protects teeth from dental caries, the most common chronic disease worldwide. In addition to a high mineral content, tooth enamel comprises organic material that is important for mechanical performance and influences the initiation and progression of caries; however, the protein composition of tooth enamel has not been fully characterized. Here, we determined that epithelial hair keratins, which are crucial for maintaining the integrity of the sheaths that support the hair shaft, are expressed in the enamel organ and are essential organic components of mature enamel. Using genetic and intraoral examination data from 386 children and 706 adults, we found that individuals harboring known hair disorder-associated polymorphisms in the gene encoding keratin 75 (KRT75), KRT75(A161T) and KRT75(E337K), are prone to increased dental caries. Analysis of teeth from individuals carrying the KRT75(A161T) variant revealed an altered enamel structure and a marked reduction of enamel hardness, suggesting that a functional keratin network is required for the mechanical stability of tooth enamel. Taken together, our results identify a genetic locus that influences enamel structure and establish a connection between hair disorders and susceptibility to dental caries.

Mouse models have greatly helped in elucidating the molecular mechanisms involved in hair formation and regeneration. Recent publications have reviewed the genes involved in mouse hair development based on the phenotype of transgenic, knockout and mutant animal models. While much of this information has been instrumental in determining molecular aspects of human hair development and cycling, mice exhibit a specific pattern of hair morphogenesis and hair distribution throughout the body that cannot be directly correlated to human hair. In this mini-review, we discuss specific aspects of human hair follicle development and present an up-to-date summary of human genetic disorders associated with abnormalities in hair follicle morphogenesis, structure or regeneration., (Published by Elsevier Ltd.)

Mutations in DLX3 in humans lead to defects in craniofacial and appendicular bones, yet the in vivo activities related to Dlx3 function during normal skeletal development have not been fully elucidated. Here we used a conditional knockout approach to analyze the effects of neural crest deletion of Dlx3 on craniofacial bones development. At birth, mutant mice exhibit a normal overall positioning of the skull bones, but a change in the shape of the calvaria was observed. Molecular analysis of the genes affected in the frontal bones and mandibles from these mice identified several bone markers known to affect bone development, with a strong prediction for increased bone formation and mineralization in vivo. Interestingly, while a subset of these genes were similarly affected in frontal bones and mandibles (Sost, Mepe, Bglap, Alp, Ibsp, Agt), several genes, including Lect1 and Calca, were specifically affected in frontal bones. Consistent with these molecular alterations, cells isolated from the frontal bone of mutant mice exhibited increased differentiation and mineralization capacities ex vivo, supporting cell autonomous defects in neural crest cells. However, adult mutant animals exhibited decreased bone mineral density in both mandibles and calvaria, as well as a significant increase in bone porosity. Together, these observations suggest that mature osteoblasts in the adult respond to signals that regulate adult bone mass and remodeling. This study provides new downstream targets for Dlx3 in craniofacial bone, and gives additional evidence of the complex regulation of bone formation and homeostasis in the adult skeleton., (Copyright © 2012 Wiley Periodicals, Inc.)

During development, Dlx3 is expressed in ectodermal appendages such as hair and teeth. Thus far, the evidence that Dlx3 plays a crucial role in tooth development comes from reports showing that autosomal dominant mutations in DLX3 result in severe enamel and dentin defects leading to abscesses and infections. However, the normal function of DLX3 in odontogenesis remains unknown. Here, we use a mouse model to demonstrate that the absence of Dlx3 in the neural crest results in major impairment of odontoblast differentiation and dentin production. Mutant mice develop brittle teeth with hypoplastic dentin and molars with an enlarged pulp chamber and underdeveloped roots. Using this mouse model, we found that dentin sialophosphoprotein (Dspp), a major component of the dentin matrix, is strongly down-regulated in odontoblasts lacking Dlx3. Using ChIP-seq, we further demonstrate the direct binding of Dlx3 to the Dspp promoter in vivo. Luciferase reporter assays determined that Dlx3 positively regulates Dspp expression. This establishes a regulatory pathway where the transcription factor Dlx3 is essential in dentin formation by directly regulating a crucial matrix protein.

Small ubiquitin-like modifiers (SUMO) are post-translational modifiers that regulate target protein activity in diverse ways. The most common group of SUMO substrates is transcription factors, whose transcriptional activity can be altered positively or negatively as a result of SUMOylation. DLX3 is a homeodomain transcription factor involved in placental development, in the differentiation of structures involving epithelial-mesenchymal interactions, such as hair, teeth and nails, and in bone mineralization. We identified two potential SUMOylation sites in the N-terminal domain of DLX3 at positions K83 and K112. Among the six members of the Distal-less family, DLX3 is the only member containing these sites, which are highly conserved among vertebrates. Co-expression experiments demonstrated that DLX3 can be SUMOylated by SUMO1. Site-directed mutagenesis of lysines 83 and 112 to arginines (K83R and K112R) demonstrated that only K112 is involved in SUMOylation. Immunocytochemical analysis determined that SUMOylation does not affect DLX3 translocation to the nucleus and favors perinuclear localization. Moreover, using electrophoresis mobility shift assay (EMSA), we found that DLX3 is still able to bind DNA when SUMOylated. Using luciferase reporter assays, we showed that DLX3(K112R) exhibits a significantly lower transcriptional activity compared to DLX3(WT), suggesting that SUMOylation has a positive effect on DLX3 activity. We identified a new level of regulation in the activity of DLX3 that may play a crucial role in the regulation of hair, teeth, and bone development., (Copyright © 2010 Wiley-Liss, Inc.)

An intriguing question in developmental biology is how epidermal pattern formation processes are established and what are the molecular mechanisms involved in these events. The establishment of the pattern is concomitant with the formation of ectodermal appendages, which involves complex interactions between the epithelium and the underlying mesenchyme. Among ectodermal appendages, hair follicles are the "mini organs" that produce hair shafts. Several developmental and structural features are common to all hair follicles and to the hair shaft they produce. However, many different hair types are produced in a single organism. Also, different characteristics can be observed depending on the part of the body where the hair follicle is formed. Here, we review the mechanisms involved in the patterning of different hair types during mouse embryonic development as well as the influence of the body axes on hair patterning.

The epidermis is a stratified epithelium which develops depending on the transcription factor p63, a member of the p53 family of transcription factors. p63 is strongly expressed in the innermost basal layer where highly proliferative epithelial cells reside. p63 functions as a molecular switch that initiates epithelial stratification or cell fate determination while regulating proliferation and differentiation of developmentally mature keratinocytes. p63 acts upstream of Dlx3 homeobox gene in a transcriptional regulatory pathway relevant to ectodermal dysplasia. Here we show that Dlx3 triggers p63 protein degradation by a proteasome-dependent pathway. Mutant DeltaNp63alpha in which Threonine397 and Serine383 were replaced with Alanine as well as C-terminal truncated versions of DeltaNp63alpha are resistant to Dlx3-mediated degradation. Transient expression of Dlx3 is associated with Raf1 phosphorylation. Dlx3 is unable to promote p63 degradation in Raf1 depleted MEF cells or upon pharmacological knockdown of Raf1. Our data support a previously unrecognized role for Dlx3 in posttranslational regulation of DeltaNp63alpha protein level, a mechanism that may contribute to reduce the abundance of DeltaNp63alpha during differentiation of stratified epithelia.

Homeobox genes are an evolutionarily conserved class of transcription factors that are key regulators during developmental processes such as regional specification, patterning, and differentiation. In this review, we summarize the expression pattern, loss- and/or gain-of-function mouse models, and naturally occurring mouse and human mutations of known homeobox genes required for the development of ectodermal appendages., ((c) 2008 Wiley-Liss, Inc.)

The homeodomain protein Distal-less-3 (Dlx3) plays a crucial role during embryonic development. This transcription factor is known to be essential for placental formation and to be involved in skin and skeletal organogenesis. In humans, a frameshift mutation in the coding sequence of the DLX3 gene results in an ectodermal dysplasia called Tricho-Dento-Osseous syndrome (TDO). The main features of this autosomal dominant disorder are defects in hair, teeth, and bone. To investigate the functional alterations caused by the mutated DLX3(TDO) isoform ex vivo, we used tetracycline-inducible osteoblastic and keratinocyte cell lines and calvarial derived osteoblasts in which the expression of DLX3(WT) and/or DLX3(TDO) could be regulated and monitored. Immunocytochemical analysis revealed that both DLX3(WT) and DLX3(TDO) recombinant proteins are targeted to the nucleus. However, as demonstrated by electrophoresis mobility shift assay, DLX3(TDO) is not able to bind to the canonical Dlx3 binding site. Furthermore, we demonstrate that the frameshifted C-terminal domain in DLX3(TDO) is accountable for the loss of DNA binding activity because the C-terminal domain in DLX3(WT) is not required for DNA binding activity. Although DLX3(TDO) alone cannot bind to a Dlx3 responsive element, when DLX3(WT) and DLX3(TDO) are co-expressed they form a complex that can bind DNA. Concomitant with the inability to bind DNA, DLX3(TDO) has a defective transcriptional activity. Moreover, the transcriptional activity of DLX3(WT) is significantly reduced in the presence of the mutated isoform, indicating that DLX3(TDO) has a dominant negative effect on DLX3(WT) transcriptional activity.

Heat shock protein (Hsp) 25 is a member of the small Hsp family. High levels of Hsp25 can be detected in skin. During adult epidermis differentiation, the concentration of Hsp25 increases as the distance of keratinocytes from the basal layer increases, in parallel with the extent of keratinization. We previously showed that Hsp25, mouse keratin (MK) 5, and MK14 participated in the formation of characteristic ring-shaped aggregates during the differentiation of the PAM212 keratinocyte cell line. We suggested that Hsp25 was involved in the disorganization of the MK5-MK14 keratin network before the establishment of the MK1-MK10 keratin network at the beginning of epidermis stratification. In this study, we have investigated the distribution of Hsp25 and keratins throughout skin development. We demonstrate that the distribution of Hsp25 and MK5 in the epidermis at the beginning of stratification and before keratinization is similar to that observed in PAM212 keratinocytes. These results indicate that there is a strong correlation between the mechanism we described ex vivo and the events taking place in vivo. Moreover, we show that Hsp25 is produced in different cell types in the epidermis and in the hair follicle at different stages of their development. Thus, our results suggest that Hsp25 is involved in more than one process during skin development.

HSP25 is a member of the small heat shock protein family. This 25-kDa protein exhibits a highly specific distribution during mouse embryonic development. Although multiple functions have been proposed for HSP25, the role it plays during differentiation is still unknown. High levels of HSP25 can be detected in embryonic and adult skin. During epidermis differentiation, the concentration of HSP25 increases with the distance of keratinocytes from the basal layer, in parallel with the extent of keratinization. We used an ex vivo cellular system, PAM212 cells, to analyze quantitatively and qualitatively the dynamics of HSP25 production and phosphorylation during the differentiation of keratinocytes. Our observations suggest that HSP25 is involved in two steps of PAM212 keratinocyte differentiation. Shortly after the induction of differentiation, a transient hyperphosphorylation of HSP25 seems to be essential for the expression of differentiation markers. Later, the chaperone-active form of HSP25 is organized progressively into characteristic aggregates involved in the dynamics of keratin filament networks.