Satrajit Sinha

Inhibition of Oral Mucosal Cell Wound Healing by Bisphosphonates

PURPOSE Bisphosphonates (BPs) are a widely used class of drugs that are effective in the treatment and prevention of osteoporosis, hypercalcemia of malignancy, and bone metastases associated with multiple myeloma, breast cancer, and other solid tumors. In the past several years there have been numerous reports describing the occurrence of osteonecrosis of the jaws (ONJ) associated with these drugs. Whether the ONJ lesion initiates in the oral mucosa or derives from the underlying bone is not well understood. In this report we describe the effect of pamidronate, a second-generation BP, on oral mucosal cells. MATERIALS AND METHODS Murine oral keratinocytes were isolated and exposed to pamidronate at a range of clinically relevant doses. Cellular proliferation was measured using a MTS/PMS reagent-based kit and wound healing was examined with a scratch assay. To determine whether oral keratinocytes undergo apoptosis following exposure to pamidronate, TUNEL, caspase-3, and DAPI apoptosis assays were performed. RESULTS We show that BP pretreatment of oral mucosal cells inhibits proliferation and wound healing at clinically relevant doses, and that this inhibition is not due to cellular apoptosis. CONCLUSIONS To our knowledge this is the first report investigating the effect of nitrogen-containing BPs on oral mucosal cells. This study suggests that BPs inhibit oral keratinocyte wound healing which may play a significant role in the initiation of ONJ.

TAp63 Prevents Premature Aging by Promoting Adult Stem Cell Maintenance

The cellular mechanisms that regulate the maintenance of adult tissue stem cells are still largely unknown. We show here that the p53 family member, TAp63, is essential for maintenance of epidermal and dermal precursors and that, in its absence, these precursors senesce and skin ages prematurely. Specifically, we have developed a TAp63 conditional knockout mouse and used it to ablate TAp63 in the germline (TAp63(-/-)) or in K14-expressing cells in the basal layer of the epidermis (TAp63(fl/fl);K14cre+). TAp63(-/-) mice age prematurely and develop blisters, skin ulcerations, senescence of hair follicle-associated dermal and epidermal cells, and decreased hair morphogenesis. These phenotypes are likely due to loss of TAp63 in dermal and epidermal precursors since both cell types show defective proliferation, early senescence, and genomic instability. These data indicate that TAp63 serves to maintain adult skin stem cells by regulating cellular senescence and genomic stability, thereby preventing premature tissue aging.

ΔNp63 knockout mice reveal its indispensable role as a master regulator of epithelial development and differentiation

The transcription factor p63 is important in the development of the skin as p63-null mice exhibit striking defects in embryonic epidermal morphogenesis. Understanding the mechanisms that underlie this phenotype is complicated by the existence of multiple p63 isoforms, including TAp63 and ΔNp63. To investigate the role of ΔNp63 in epidermal morphogenesis we generated ΔNp63 knock-in mice in which the ΔNp63-specific exon is replaced by GFP. Homozygous ΔNp63gfp/gfp animals exhibit severe developmental anomalies including truncated forelimbs and the absence of hind limbs, largely phenocopying existing knockouts in which all p63 isoforms are deleted. ΔNp63-null animals show a poorly developed stratified epidermis comprising isolated clusters of disorganized epithelial cells. Despite the failure to develop a mature stratified epidermis, the patches of ΔNp63-null keratinocytes are able to stratify and undergo a program of terminal differentiation. However, we observe premature expression of markers associated with terminal differentiation, which is unique to ΔNp63-null animals and not evident in the skin of mice lacking all p63 isoforms. We posit that the dysregulated and accelerated keratinocyte differentiation phenotype is driven by significant alterations in the expression of key components of the Notch signaling pathway, some of which are direct transcriptional targets of ΔNp63 as demonstrated by ChIP experiments. The analysis of ΔNp63gfp/gfp knockout mice reaffirms the indispensable role of the ΔN isoform of p63 in epithelial biology and confirms that ΔNp63-null keratinocytes are capable of committing to an epidermal cell lineage, but are likely to suffer from diminished renewal capacity and an altered differentiation fate.

Three classes of mutations in the A subunit of the CCAAT-binding factor CBF delineate functional domains involved in the three-step assembly of the CBF-DNA complex.

The mammalian CCAAT-binding factor CBF (also called NF-Y or CP1) consists of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and present in the CBF-DNA complex. In this study we first established the stoichiometries of the CBF subunits, both in the CBF molecule and in the CBF-DNA complex, and showed that one molecule of each subunit is present in the complex. To begin to understand the interactions between the CBF subunits and DNA, we performed a mutational analysis of the CBF-A subunit. This analysis identified three classes of mutations in the segment of CBF-A that is conserved in Saccharomyces cerevisiae and mammals. Analysis of the first class of mutants revealed that a major part of the conserved segment was essential for interactions with CBF-C to form a heterodimeric CBF-A/CBF-C complex. The second class of mutants identified a segment of CBF-A that is necessary for interactions between the CBF-A/CBF-C heterodimer and CBF-B to form a CBF heterotrimer. The third class defined a domain of CBF-A involved in binding the CBF heterotrimer to DNA. The second and third classes of mutants acted as dominant negative mutants inhibiting the formation of a complex between the wild-type CBF subunits and DNA. The segment of CBF-A necessary for DNA binding showed sequence homology to a segment of CBF-C. Interestingly, these sequences in CBF-A and CBF-C were also homologous to the sequences in the histone-fold motifs of histones H2B and H2A, respectively, and to the archaebacterial histone-like protein HMf-2. We discuss the functional domains of CBF-A and the properties of CBF in light of these sequence homologies and propose that an ancient histone-like motif in two CBF subunits controls the formation of a heterodimer between these subunits and the assembly of a sequence-specific DNA-protein complex.

Determination of functional domains in the C subunit of the CCAAT- binding factor (CBF) necessary for formation of a CBF-DNA complex: CBF-B interacts simultaneously with both the CBF-A and CBF-C subunits to form a heterotrimeric CBF molecule

The mammalian CCAAT-binding factor (CBF; also called NF-Y and CP1) is a heterotrimeric protein consisting of three subunits, CBF-A, CBF-B, and CBF-C, all of which are required for DNA binding and all of which are present in the CBF-DNA complex. In this study using cross-linking and immunoprecipitation methods, we first established that CBF-B interacts simultaneously with both subunits of the CBF-A-CBF-C heterodimer to form a heterotrimeric CBF molecule. We then performed a mutational analysis of CBF-C to define functional interactions with the other two CBF subunits and with DNA using several in vitro assays and an in vivo yeast two-hybrid system. Our experiments established that the evolutionarily conserved segment of CBF-C, which shows similarities with the histone-fold motif of histone H2A, was necessary for formation of the CBF-DNA complex. The domain of CBF-C which interacts with CBF-A included a large portion of this segment, one that corresponds to the segment of the histone-fold motif in H2A used for interaction with H2B. Two classes of interactions involved in formation of the CBF-A-CBF-C heterodimer were detected; one class, provided by residues in the middle of the interaction domain, was needed for formation of the CBF-A-CBF-C heterodimer. The other, provided by sequences flanking those of the first class was needed for stabilization of the heterodimer. Two separate domains were identified in the conserved segment of CBF-C for interaction with CBF-B; these were located on each side of the CBF-A interaction domain. Since our previous experiments identified a single CBF-B interaction domain in the histone-fold motif of CBF-A, we propose that a tridentate interaction domain in the CBF-A-CBF-C heterodimer interacts with the 21-amino-acid-long subunit interaction domain of CBF-B. Together with our previous mutational analysis of CBF-A (S. Sinha, I.-S. Kim, K.-Y. Sohn, B. de Crombrugghe, and S. N. Maity, Mol. Cell. Biol. 16:328-337, 1996), this study demonstrates that the histone fold-motifs of CBF-A and CBF-C interact with each other to form the CBF-A-CBF-C heterodimer and generate a hybrid surface which then interacts with CBF-B to form the heterotrimeric CBF molecule.

Hdac1 and Hdac2 act redundantly to control p63 and p53 functions in epidermal progenitor cells

Epidermal and hair follicle development from surface ectodermal progenitor cells requires coordinated changes in gene expression. Histone deacetylases alter gene expression programs through modification of chromatin and transcription factors. We find that deletion of ectodermal Hdac1 and Hdac2 results in dramatic failure of hair follicle specification and epidermal proliferation and stratification, phenocopying loss of the key ectodermal transcription factor p63. Although expression of p63 and its positively regulated basal cell targets is maintained in Hdac1/2-deficient ectoderm, targets of p63-mediated repression, including p21, 14-3-3σ, and p16/INK4a, are ectopically expressed, and HDACs bind and are active at their promoter regions in normal undifferentiated keratinocytes. Mutant embryos display increased levels of acetylated p53, which opposes p63 functions, and p53 is required for HDAC inhibitor-mediated p21 expression in keratinocytes. Our data identify critical requirements for HDAC1/2 in epidermal development and indicate that HDAC1/2 directly mediate repressive functions of p63 and suppress p53 activity.

Elf5 inhibits the epithelial–mesenchymal transition in mammary gland development and breast cancer metastasis by transcriptionally repressing Snail2

The epithelial–mesenchymal transition (EMT) is a complex process that occurs during organogenesis and in cancer metastasis. Despite recent progress, the molecular pathways connecting the physiological and pathological functions of EMT need to be better defined. Here we show that the transcription factor Elf5, a key regulator of mammary gland alveologenesis, controls EMT in both mammary gland development and metastasis. We uncovered this role for Elf5 through analyses of Elf5 conditional knockout animals, various in vitro and in vivo models of EMT and metastasis, an MMTV-neu transgenic model of mammary tumour progression and clinical breast cancer samples. Furthermore, we demonstrate that Elf5 suppresses EMT by directly repressing the transcription of Snail2, a master regulator of mammary stem cells and a known inducer of EMT. These findings establish Elf5 not only as a key cell lineage regulator during normal mammary gland development, but also as a suppressor of EMT and metastasis in breast cancer.

Connexin 26 regulates epidermal barrier and wound remodeling and promotes psoriasiform response

Inflammatory skin disorders result in significant epidermal changes, including keratinocyte hyperproliferation, incomplete differentiation, and impaired barrier. Here we test whether, conversely, an impaired epidermal barrier can promote an inflammatory response. Mice lacking the transcription factor Kruppel-like factor 4 (Klf4) have a severe defect in epidermal barrier acquisition. Transcription profiling of Klf4(-/-) newborn skin revealed similar changes in gene expression to involved psoriatic plaques, including a significant upregulation of the gap junction protein connexin 26 (Cx26). Ectopic expression of Cx26 from the epidermis-specific involucrin (INV) promoter (INV-Cx26) demonstrated that downregulation of Cx26 is required for barrier acquisition during development. In juvenile and adult mice, persistent Cx26 expression kept wounded epidermis in a hyperproliferative state, blocked the transition to remodeling, and led to an infiltration of immune cells. Mechanistically, ectopic expression of Cx26 in keratinocytes resulted in increased ATP release, which delayed epidermal barrier recovery and promoted an inflammatory response in resident immune cells. These results provide a molecular link between barrier acquisition in utero and epidermal remodeling after wounding. More generally, these studies suggest that the most effective treatments for inflammatory skin disorders might concomitantly suppress the immune response and enhance epidermal differentiation to restore the barrier.

Elf5 conditional knockout mice reveal its role as a master regulator in mammary alveolar development: Failure of Stat5 activation and functional differentiation in the absence of Elf5

The transcription factor Elf5 plays an important role in mammary gland development. However, because of the embryonic lethality of Elf5 straight knockout mice, prior studies have been limited to experiments with Elf5 haploinsufficient animals, overexpression systems or transplants. Here, we have utilized K14-Cre to generate mammary-gland specific Elf5 conditional knockout mice. During pregnancy, Elf5-null mammary epithelium completely failed to initiate alveologenesis, and a characteristic of virgin ductal epithelial cells persisted postpartum. We demonstrate that the loss of Elf5 leads to the absence of alveolar secretory markers confirming previous published data. Interestingly, the developmental block due to a lack of Elf5 could not be restored by multiple gestations. Elf5-null mammary epithelial cells also display disorganized cell structures as evident by altered cell polarities, which might be the cause for collapsed lumina. We observe reduced levels of Stat5 and attenuated Stat5 activity as measured by p-Stat5 levels both in Elf5-null mammary glands as well as cultured mammary epithelial cells. This data suggests that the failure of alveolar and lactogenic differentiation due to the loss of Elf5 is mediated in part due to impaired Stat5 activity. In support of this hypothesis, we show by ChIP experiments that Stat5a promoter contains a conserved Elf5-binding site that is occupied by Elf5 in mammary glands. Mammary epithelia lacking Elf5 exhibited downregulation of several other critical genes involved in alveologenesis, suggesting Elf5 as a master regulator in alveolar development. We propose a model for Elf5-mediated alveolar development, in which Elf5 regulates the expression of key mediators of the PrlR/Jak2/Stat5 signaling pathway.

Defining the regulatory factors required for epidermal gene expression.

ABSTRACT Keratins K5 and K14 are the hallmarks of mitotically active keratinocytes of stratified epithelia. They are transcribed at a high level and in a tissue-specific manner, enabling us to use the K14 gene to elucidate the regulatory mechanism underlying epidermis-specific transcription. We have identified four DNase I-hypersensitive sites (HSs) present in the 5′ regulatory sequences of the K14 gene under specific conditions where the gene is actively expressed. Two of these sites (HSsII and -III) are conserved in position and sequence within the human and mouse K14 genes. Using an in vivo transgenic approach and an in vitro keratinocyte culture approach, we have discovered that most of K14s transcriptional activity is restricted to a novel 700-bp regulatory domain encompassing these HSs. This enhancer is sufficient to confer epidermis-specific activity to a heterologous promoter in transfection assays in culture and in transgenic mice in vivo. A 125-bp DNA fragment encompassing HSsII harbors the majority of the transactivation activity in vitro, and electrophoretic mobility shift and mutational assays reveal a role for AP-1, ets, and AP-2 family members in orchestrating the keratinocyte-preferred expression of HSsII. The HSsII element also confers epidermal expressivity to a heterologous promoter in transgenic mice, although it is not sufficient on its own to fully restrict activity to keratinocytes. Within the HSsII element, the ets and AP-2 sites appear to be most critical in collaborating to regulate epidermal specificity in vivo.