IU Indianapolis ScholarWorks :: Browsing by Author "Spandau, Dan F."

Browsing by Author "Spandau, Dan F."

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Creatine and Nicotinamide Prevent Oxidant-Induced Senescence in Human Fibroblasts
(MDPI, 2021-11-16) Mahajan, Avinash S.; Arikatla, Venkata S.; Thyagarajan, Anita; Zhelay, Tetyana; Sahu, Ravi P.; Kemp, Michael G.; Spandau, Dan F.; Travers, Jeffrey B.; Dermatology, School of Medicine
Dermal fibroblasts provide structural support by producing collagen and other structural/support proteins beneath the epidermis. Fibroblasts also produce insulin-like growth factor-1 (IGF-1), which binds to the IGF-1 receptors (IGF-1Rs) on keratinocytes to activate signaling pathways that regulate cell proliferation and cellular responses to genotoxic stressors like ultraviolet B radiation. Our group has determined that the lack of IGF-1 expression due to fibroblast senescence in the dermis of geriatric individuals is correlated with an increased incidence of skin cancer. The present studies tested the hypothesis that pro-energetics creatine monohydrate (Cr) and nicotinamide (NAM) can protect normal dermal human fibroblasts (DHF) against experimentally induced senescence. To that end, we used an experimental model of senescence in which primary DHF are treated with hydrogen peroxide (H2O2) in vitro, with senescence measured by staining for beta-galactosidase activity, p21 protein expression, and senescence associated secretory phenotype cytokine mRNA levels. We also determined the effect of H2O2 on IGF-1 mRNA and protein expression. Our studies indicate that pretreatment with Cr or NAM protects DHF from the H2O2-induced cell senescence. Treatment with pro-energetics post-H2O2 had no effect. Moreover, these agents also inhibited reactive oxygen species generation from H2O2 treatment. These studies suggest a potential strategy for protecting fibroblasts in geriatric skin from undergoing stress-induced senescence, which may maintain IGF-1 levels and therefore limit carcinogenesis in epidermal keratinocytes.
The eIF2 kinase GCN2 directs keratinocyte collective cell migration during wound healing via coordination of reactive oxygen species and amino acids
(American Society for Biochemistry and Molecular Biology, 2021-11) Miles, Rebecca R.; Amin, Parth H.; Diaz, Miguel Barriera; Misra, Jagannath; Aukerman, Erica; Das, Amitava; Ghosh, Nandini; Guith, Tanner; Knierman, Michael D.; Roy, Sashwati; Spandau, Dan F.; Wek, Ronald C.; Biochemistry and Molecular Biology, School of Medicine
Healing of cutaneous wounds requires the collective migration of epithelial keratinocytes to seal the wound bed from the environment. However, the signaling events that coordinate this collective migration are unclear. In this report, we address the role of phosphorylation of eukaryotic initiation factor 2 (eIF2) and attendant gene expression during wound healing. Wounding of human keratinocyte monolayers in vitro led to the rapid activation of the eIF2 kinase GCN2. We determined that deletion or pharmacological inhibition of GCN2 significantly delayed collective cell migration and wound closure. Global transcriptomic, biochemical, and cellular analyses indicated that GCN2 is necessary for maintenance of intracellular free amino acids, particularly cysteine, as well as coordination of RAC1-GTP-driven reactive oxygen species (ROS) generation, lamellipodia formation, and focal adhesion dynamics following keratinocyte wounding. In vivo experiments using mice deficient for GCN2 validated the role of the eIF2 kinase during wound healing in intact skin. These results indicate that GCN2 is critical for appropriate induction of collective cell migration and plays a critical role in coordinating the re-epithelialization of cutaneous wounds.
Estrogen modulates mesenchyme-epidermis interactions in the adult nipple
(Company of Biologists, 2017-04-15) Wu, Hsing-Jung; Oh, Ji Won; Spandau, Dan F.; Tholpady, Sunil; Diaz, Jesus, III; Schroeder, Laura J.; Offutt, Carlos D.; Glick, Adam B.; Plikus, Maksim V.; Koyama, Sachiko; Foley, John; Medicine, School of Medicine
Maintenance of specialized epidermis requires signals from the underlying mesenchyme; however, the specific pathways involved remain to be identified. By recombining cells from the ventral skin of the K14-PTHrP transgenic mice [which overexpress parathyroid hormone-related protein (PTHrP) in their developing epidermis and mammary glands] with those from wild type, we show that transgenic stroma is sufficient to reprogram wild-type keratinocytes into nipple-like epidermis. To identify candidate nipple-specific signaling factors, we compared gene expression signatures of sorted Pdgfrα-positive ventral K14-PTHrP and wild-type fibroblasts, identifying differentially expressed transcripts that are involved in WNT, HGF, TGFβ, IGF, BMP, FGF and estrogen signaling. Considering that some of the growth factor pathways are targets for estrogen regulation, we examined the upstream role of this hormone in maintaining the nipple. Ablation of estrogen signaling through ovariectomy produced nipples with abnormally thin epidermis, and we identified TGFβ as a negatively regulated target of estrogen signaling. Estrogen treatment represses Tgfβ1 at the transcript and protein levels in K14-PTHrP fibroblasts in vitro, while ovariectomy increases Tgfb1 levels in K14-PTHrP ventral skin. Moreover, ectopic delivery of Tgfβ1 protein into nipple connective tissue reduced epidermal proliferation. Taken together, these results show that specialized nipple epidermis is maintained by estrogen-induced repression of TGFβ signaling in the local fibroblasts.
Estrogen receptor involvement in the response of human keratinocytes to ultraviolet B irradiation
(2014) Farrington, Daphne L.; Spandau, Dan F.; Harrington, Maureen A.; Nakshatri, Harikrishna
The signaling mechanisms involved in UVB-induced skin cancer are complex and although the scope of this work is inherently limited in focus, the findings may provide insight into how estrogen receptor signaling impacts cell growth, senescence, and apoptosis to protect keratinocytes. Additional signaling due to E2-activation of the estrogen receptor may provide back-up or redundant pathways in response to UVB.
Ex vivo culture of mouse skin activates an interleukin 1 alpha-dependent inflammatory response
(Wiley, 2020-01) Zhou, Hong-Ming; Slominski, Radomir M.; Seymour, Leroy J.; Bell, Maria C.; Dave, Priya; Atumonye, Joseph; Wright, William, III.; Dawes, Avery; Griesenauer, Brad; Paczesny, Sophie; Kaplan, Mark H.; Spandau, Dan F.; Turner, Matthew J.; Dermatology, School of Medicine
Ex vivo culture of mouse and human skin causes an inflammatory response characterized by production of multiple cytokines. We used ex vivo culture of mouse tail skin specimens to investigate mechanisms of this skin culture-induced inflammatory response. Multiplex assays revealed production of interleukin 1 alpha (IL-1α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), chemokine C-X-C motif ligand 1 (CXCL1), granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) during skin culture, and quantitative PCR revealed transcripts for these proteins were also increased. Ex vivo cultures of skin from myeloid differentiation primary response 88 deficient mice (Myd88-/- ) demonstrated significantly reduced expression of transcripts for the aforementioned cytokines. The same result was observed with skin from interleukin 1 receptor type 1 deficient mice (Il1r1-/- ). These data suggested the IL-1R1/MyD88 axis is required for the skin culture-induced inflammatory response and led us to investigate the role of IL-1α and IL-1β (the ligands for IL-1R1) in this process. Addition of IL-1α neutralizing antibody to skin cultures significantly reduced expression of Cxcl1, Il6 and Csf3. IL-1β neutralization did not reduce levels of these transcripts. These studies suggest that IL-1α promotes the skin the culture-induced inflammatory response.
Expansion of specialized epidermis induced by hormonal state and mechanical strain
(Elsevier, 2015-05) Wu, Hsin-Jung; Easwaran, Teresa; Offutt, Carlos D.; Elgar, Richard Levi; Spandau, Dan F.; Koyama, Sachiko; Foley, John; Department of Medicine, IU School of Medicine
In mammals, some sites of specialized skin such as the palms, soles, and lips grow proportionally with the animal. However, other types of specialized skin such as the nipple and anal/genital region are dramatically altered with changes of reproductive status. The specific cell types that mediate the growth of these sites have not been identified. In the mouse, we observed a dramatic expansion of the specialized epidermis of the nipple, coupled to changes in connective tissue and hair shaft density, which we designate as areola formation. During this process thymidine analog uptake was elevated in the epidermis and hair follicles. Although there were no changes in connective tissue cell proliferation, we did observe an altered expression of extracellular matrix genes. In addition, the fibroblasts of the virgin nipple areola and region showed increased transcript and protein levels for estrogen, progesterone, relaxin, and oxytocin relative to those of ventral skin. To determine the role of pregnancy, lactation hormonal milieu, and localized mechanical strain on areola formation, we created models that separated these stimuli and evaluated changes in gross structure, proliferation and protein expression. While modest increases of epidermal proliferation and remodeling of connective tissue occurred as a result of individual stimuli, areola formation required exposure to pregnancy hormones, as well as mechanical strain.
GCN2 EIF2 Kinase is Critical for Keratinocyte Collective Migration and Wound Healing
(2021-12) Miles, Rebecca Ruth; Wek, Ronald C.; Spandau, Dan F.; Kaplan, Mark H.; Turner, Matthew J.; Heuer, Josef G.
A critical factor in the healing of a cutaneous wound is the closure of the wound bed that is accomplished by collectively migrating keratinocytes. Wounds that fail to heal appropriately are a significant burden on patient well-being as well as healthcare systems. Further understanding of the molecular pathways involved in wound healing is needed to find new treatments to accelerate healing and reduce treatment costs. Previously, we demonstrated that the integrated stress response (ISR) is critical for keratinocyte response to multiple stresses. Because wounding and repair mechanisms can induce stresses in the skin, we hypothesized that the ISR plays a central role in wound healing. The ISR features a family of stress-activated protein kinases phosphorylate the translation factor eIF2 (eIF2α-P), resulting in diminished global protein synthesis coincident with preferential translation of gene transcripts that lead to the remedy of the stress. Wounding of immortalized NTERT human keratinocyte monolayers led to rapid activation of the eIF2 kinase GCN2, and subsequent eIF2α-P and translational control. Deletion of GCN2 in wounding assays diminished eIF2α-P and translational control during wound healing. Global transcriptome analysis of wounded keratinocytes revealed that deletion of GCN2 induced a compensatory unfolded protein response and dysregulation of mRNAs important for cellular migration. Pathway analysis suggested that GCN2 is necessary for proper activation of key signaling networks and subsequent coordination of RAC-GTP driven reactive oxygen species (ROS) generation following wounding. Additionally, amino acid control of cysteine is regulated by GCN2. We therefore investigated ROS levels following wounding and observed that GCN2 was required for proper ROS induction and actin reorganization in leading edge keratinocytes and that these changes were coincident with reduced RAC and RHO activation and cysteine depletion. The loss of leading-edge ROS in GCN2-deleted cells can be phenocopied with NOX inhibition. Lastly, mice deleted for GCN2 exhibited delayed wound healing compared to WT controls in an excisional wound healing model. These results indicate that GCN2 is required for the induction of collective cell migration and plays a critical role in coordinating the re-epithelialization of cutaneous wounds. We propose the ISR is a potential therapeutic target in chronic wounds.
Human keratinocyte differentiation requires translational control by the eIF2α kinase GCN2
(Elsevier, 2017) Collier, Ann E.; Wek, Ronald C.; Spandau, Dan F.; Department of Dermatology, IU School of Medicine
Appropriate and sequential differentiation of keratinocytes is essential for all functions of the human epidermis. While transcriptional regulation has proven to be important for keratinocyte differentiation, little is known about the role of translational control. A key mechanism for modulating translation is through phosphorylation of the α subunit of eIF2. A family of different eIF2α kinases function in the integrative stress response to inhibit general protein synthesis coincident with preferential translation of select mRNAs that participate in stress alleviation. Here we demonstrate that translational control through eIF2α phosphorylation is required for normal keratinocyte differentiation. Analyses of polysome profiles revealed that key differentiation genes, including involucrin, are bound to heavy polysomes during differentiation, despite decreased general protein synthesis. Induced eIF2α phosphorylation by the GCN2 protein kinase facilitated translational control and differentiation-specific protein expression during keratinocyte differentiation. Furthermore, loss of GCN2 thwarted translational control, normal epidermal differentiation, and differentiation gene expression in organotypic skin culture. These findings underscore a previously unknown function for GCN2 phosphorylation of eIF2α and translational control in the formation of an intact human epidermis.
Human keratinocytes utilize the integrated stress response to adapt to environmental stress
(2017-06) Collier, Ann E.; Spandau, Dan F.; Wek, Ronald C.; Travers, Jeffrey B.; Turchi, John J.; Turner, Matthew J.
Human skin, consisting of the outer epidermis and inner dermis, serves as a barrier that protects the body from an onslaught of environmental stresses. Keratinocytes in the stratified epidermis undergo sequential differentiation that consists of multiple layers of cells differing in structure and function. Therefore, keratinocytes must not only combat environmental stress, but need to undergo massive changes in gene expression and morphology to form a proper barrier. One mode by which cells cope with stress and differentiation is through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α-P), which causes global inhibition of protein synthesis coincident with preferential translation of select gene transcripts. Translational repression allows stressed cells to conserve energy and prioritize pro-survival processes to alleviate stress damage. Since eIF2α kinases are each activated by distinct types of stress, this pathway is referred to as the Integrated Stress Response (ISR). We sought to identify the roles of the ISR in the keratinocyte response to the stresses associated with differentiation and ultraviolet B (UVB) irradiation. In this thesis, we show that both general and gene-specific translational control in the ISR are activated following differentiation or UVB irradiation of human keratinocytes. ISR deficiency through genetic modifications or pharmacological interventions caused severe divergence from the appropriate keratinocyte response to differentiation or UVB. Differentiation genes were selectively translated by eIF2α-P, and inhibition of the ISR diminished their induction during differentiation. Furthermore, loss of the eIF2α kinase GCN2 (EIF2AK4) adversely affected the ability of keratinocytes to stratify in three dimensional cultures. Our analysis also revealed a non-canonical ISR response following UVB irradiation, in which downstream factors ATF4 (CREB2) and CHOP (DDIT3/GADD153) were poorly expressed due to repressed transcription, despite preferential translation in response to eIF2α-P. The ISR was cytoprotective during UVB and we found that eIF2α-P was required for a UVB induced G1 arrest, cell fate determination, and DNA repair via a mechanism involving translational control of human CDKN1A (p21 protein) transcript variant 4 mRNA. Collectively, this thesis describes novel roles for the ISR in keratinocyte differentiation and response to UVB, emphasizing the utility of targeting translational control in skin disease therapy.
IL-4 impairs wound healing potential in the skin by repressing fibronectin expression
(Elsevier, 2017-01) Serezani, Ana PM; Bozdogan, Gunseli; Sehra, Sarita; Walsh, Daniel; Krishnamurthy, Purna; Potchanant, Elizabeth A Sierra; Nalepa, Grzegorz; Goenka, Shreevrat; Turner, Matthew J.; Spandau, Dan F.; Kaplan, Mark H.; Pediatrics, School of Medicine
BACKGROUND: Atopic dermatitis (AD) is characterized by intense pruritis and is a common childhood inflammatory disease. Many factors are known to affect AD development, including the pleiotropic cytokine IL-4. Yet little is known regarding the direct effects of IL-4 on keratinocyte function. OBJECTIVE AND METHODS: In this report RNA sequencing and functional assays were used to define the effect of the allergic environment on primary keratinocyte function and wound repair in mice. RESULTS: Acute or chronic stimulation by IL-4 modified expression of more than 1000 genes expressed in human keratinocytes that are involved in a broad spectrum of nonoverlapping functions. Among the IL-4-induced changes, repression of fibronectin critically impaired the human keratinocyte wound response. Moreover, in mouse models of spontaneous and induced AD-like lesions, there was delayed re-epithelialization. Importantly, topical treatment with fibronectin restored the epidermal repair response. CONCLUSION: Keratinocyte gene expression is critically shaped by IL-4, altering cell fate decisions, which are likely important for the clinical manifestations and pathology of allergic skin disease.

Browsing by Author "Spandau, Dan F."

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