This document summarizes three research studies on skin and keratinocytes: 1) The first study found elevated expression of osteopontin splice variants in nonmelanoma skin cancers compared to normal skin and adult keratinocytes. It also found that human adult keratinocytes expressed basal or induced levels of only two osteopontin variants. 2) The second study generated a mouse model lacking the desmoglein 1 protein and found it led to perinatal lethality and impaired skin barrier formation. Desmoglein 1 appears essential for normal epidermal morphogenesis. 3) The third study used fluorescence polarization microscopy and magnetic tweezers to investigate protein organization and force transmission at

1. 874
Elevated expression of osteopontin splice variants in nonmelanoma skin cancer
compared to normal skin and adult keratinocytes
C Chou1
, C Huang2
and P Chang3
1 Nutrition Sciences, University of Alabama at Birming-
ham, Birmingham, AL, 2 Dermatology, University of Alabama at Birmingham, Birmingham,
AL and 3 Nutrition Sciences and Dermatology, University of Alabama at Birmingham,
Birmingham, AL
Elevated expression of osteopontin (OPN), a matricellular, adhesive glycoprotein, is associated
with numerous cancer types. We have reported that ultraviolet B (UVB)-induced OPN is a
critical driver for cutaneous squamous cell carcinoma (cSCC) development in the photo-
carcinogenesis model. In human skin cancer, UVB exposure is the major risk factor on the
development of nonmelanoma skin cancer. Immunohistochemical analyses indicated that OPN
protein is markedly increased in actinic keratoses, cSCC, and differentiated basal cell carcinoma
(BCC), but not in solid basal cell epitheliomas when compared with no sun-exposed skin. This
suggests OPN may play an important role in the maintenance of the pre-malignant and ma-
lignant keratinocytes in cSCC and differentiated BCC. Whether OPN splice variants, shown to
have distinct functions, are expressed in normal and nonmelanoma skin cancer is not known.
This study assessed whether 1) OPN splice variants differ in normal skin, cSCC, and BCC, and 2)
their expression is altered in human adult keratinocytes (HaCaT) treated in vitro with UVB or 1,
25-dihydroxyvitamin D3(calcitriol), by real-time qPCR and Western blot analyses. Normal skin
expressed minimal OPN-a, OPN-b and OPN-c mRNAs. In contrast, the transcripts of OPN
variants were significantly (p<0.001) elevated in cSCC and BCC. In cSCC, each OPN isoform
expression was at similar levels. In BCC, OPN-a was significantly (p<0.05) higher than OPN-c.
Consistent with normal skin, HaCaT keratinocytes also expressed low levels of OPN-a and OPN-
b, however, OPN-c was not detected. UVB did not induce OPN expression, whereas calcitriol
stimulated OPN-a and OPN-b only. Collectively, these results indicate elevated expression of the
three OPN isoforms in nonmelanoma skin cancer compared to normal skin. Additionally, adult
keratinocytes expressed only basal and induced level of OPN-a and OPN-b, suggesting that
other cell types may be responsible for OPN-c expression.
875
A novel animal model of Desmoglein 1 (Dsg1) deficiency reveals an essential
role for Dsg1 in epidermal barrier formation
LM Godsel, GN Fitz, JL Koetsier and KJ Green Department of Pathology, Northwestern
University, Chicago, IL
Desmoglein 1 (Dsg1) is a desmosomal cadherin that first appears during skin development at
the time of periderm formation, and later is expressed in basal cells as they begin to differ-
entiate and transit into the suprabasal layers of stratified epidermis. Its importance in
epidermal function is underscored by human diseases caused by aberrant Dsg1 expression,
such as Striate Palmoplantar Keratoderma (SPPK) and SAM (Severe Skin Dermatitis, Allergies
and Metabolic Wasting). In contrast to other mouse desmosomal cadherins, Dsg1 is present as
three, tandem a, b and g genes in the cadherin cluster, which has hindered generation of a
Dsg1 model. Using CRISPR/Cas9 technology we deleted all three genes, allowing us to
investigate the importance of Dsg1 in the development and homeostasis of stratified
epidermis. Dsg1-/-
mice are born in the expected Mendelian ratios, but display a phenotype
consistent with barrier impairment and exhibit peeling, denuded skin and postnatal lethality.
Biochemical, electron and light microscopical analyses of E18.5 Dsg1-/-
skin revealed
decreased markers of differentiation, including Loricrin and Filaggrin, with no compensatory
increase in Dsg2 or Dsg3, dissociation of the stratum corneum and aberrant desmosomes with
widened intercellular spaces. Dsg1-/-
epidermis was more disorganized compared to wild-
type mice and contained enlarged keratinocytes. Further, the living epidermal layers of the
Dsg1-/-
skin were on average 10 mm thinner than wildtype littermate controls. Likewise, the
living epidermal layers of the viable adult Dsg1+/-
animals were also thinner, consistent with
the possibility that Dsg1 is required for normal epidermal morphogenesis. The Dsg1+/-
ani-
mals promise to provide a model for delineating Dsg1’s roles in epidermal morphogenesis as
well as its role in differentiation and interactions with the immune system in diseases of Dsg1
deficiency, such as SAM syndrome and SPPK.
876
Imaging nanoscale changes in desmosome protein organization
E Bartle1
, T Urner1
, T Rao1
, AP Kowalczyk2
and AL Mattheyses1
1 Department of Cell,
Developmental, and Integrative Biology, University of Alabama Birmingham, Birmingham,
AL and 2 Emory Univ Cell Biology and Dermatology Depts and Winship Cancer Institute,
Atlanta, GA
Desmosomes provide strong adhesion between epidermal keratinocytes by coupling extra-
cellular adhesions, mediated by desmosomal cadherins, to the keratin cytoskeleton. Des-
mosomes have two functional states: Ca2þ
dependent and hyper-adhesive (defined by Ca2þ
independence). We hypothesize that protein organization provides a mechanism to regulate
adhesion, and that this organization can be disrupted in disease. We are developing
microscopy approaches to study nanoscale protein organization, including fluorescence
polarization microscopy (FPM) to measure collective protein order or disorder in desmo-
somes. To probe for order, the membrane proximal EC5 domain of the cadherin desmoglein 3
was replaced with GFP (Dsg3-DEA-GFP). To induce hyper-adhesion, HaCaT cells were
treated with a protein kinase C alpha inhibitor. FPM revealed that desmosomal Dsg3-DEA-
GFP is ordered in both Ca2þ
dependent and independent cells. When Ca2þ
dependent cells
were switched to low Ca2þ
media, Dsg3-DEA-GFP became disordered and adhesion was lost.
In hyper-adhesive cells, adhesive strength and desmosomes were maintained following a
switch to low Ca2þ
media. Interestingly, in these cells there was also a loss of Dsg3-DEA-GFP
order following reduction of Ca2þ
. This result demonstrates that the rigidity of desmoglein 3 is
lost upon removal of Ca2þ
, regardless of desmosome functional state. Our results demonstrate
that cadherin trans-binding can persist after loss of tertiary structure, suggesting a central role
for signaling at the desmosomal plaque in conferring hyper-adhesion. FPM is a powerful
approach that can be applied to study desmosome structure in human disease.
877
Integration of magnetic tweezers and traction force microscopy for exploring
the mechanobiology of keratinocyte cell-cell and cell-matrix anchoring
junctions
WI Moghram1
, A Kruger2
, EA Sander1
and JC Selby3
1 Biomedical Engineering, University of
Iowa, Iowa City, IA, 2 Electrical and Computer Engineering, University of Iowa, Iowa City, IA
and 3 Dermatology and Biomedical Engineering, University of Iowa, Iowa City, IA
Recent decades have witnessed great advances in our understanding of the biophysical
mechanisms by which living cells utilize cell-cell and cell-matrix anchoring junctions to
sense and respond to perturbations in their local mechanical environment. In continuation of
these efforts, we present our preliminary work on the design, construction, and imple-
mentation of a methodology that integrates the techniques of magnetic tweezers (MT) and
traction force microscopy (TFM) to investigate open questions in the field of keratinocyte
mechanobiology. In our MT setup, we have engineered an electromagnetic needle composed
of a m-metal core with w10 mm tip, capable of generating w1 to 5 nN forces on target w2.8
mm-diameter functionalized superparamagnetic beads at bead-tip separation distances
ranging from 10 to 100 mm. Using a cascade PID control scheme for regulating magnetic flux
within our needle core, we demonstrate the ability to impose stepwise forces on target beads
with w75 ms response times in a manner that avoids force overshoot while compensating for
remnant magnetic fields that develop within the needle during use. For TFM, we employ
w150 to 600 mm-thick type I collagen gels coated with a surface layer of covalently attached
0.5 mm red-fluorescent microspheres for tracking substrate deformations. In contrast to
polyacrylamide gels that are typically used for TFM, our collagen gel substrates offer a more
physiologically relevant matrix for keratinocyte attachment, proliferation, and differentiation.
More importantly, the collagen gels are extremely compliant (E w30 to 100 Pa), and the range
of forces output from our MT device can be used to generate quantifiable substrate de-
formations. Future work will utilize this integrated methodology to explore cell-cell and cell-
matrix force transmission and mechanotransduction within multicellular epidermal con-
structs in the context of pemphigus vulgaris and bullous pemphigoid.
878
Individual variation in balance between platelet-secreted growth factors
causing contradictory effects on hair follicle could potentially impact response
to PRP therapy in patients with scalp hair loss
J Mohammed1
, M Abedin2
, R Farah1
, A Wipf3
and M Hordinsky1
1 Dermatology, University
of Minnesota, Minneapolis, MN, 2 University of Minnesota, Minneapolis, MN and 3 Uni-
versity of Minnesota, Minneapolis, MN
Platelets contain a-granules that are reservoirs of critical growth factors (GFs) regulating
cellular proliferation, migration, differentiation and angiogenesis. Due to the ability of
platelets to secrete GFs that play critical roles in the natural healing process, platelet-rich
plasma (PRP) prepared from the blood is injected to sites of injury to deliver high concen-
trations of autologous GFs. Several studies published over the past few years have evaluated
the effectiveness of PRP to treat hair loss disorders such as Androgenetic Alopecia. It is widely
believed that GFs released from platelets upon PRP injection act on skin and hair follicle stem
cells thereby promoting neovascularization and most likely, follicle differentiation. However,
despite several promising results reported from clinical trials on the therapeutic potential of
PRP, the response has been inconsistent and in some cases conflicting. Its likely that patient
response to PRP depends on the balance between platelet-secreted GFs known to promote
(PDGF-A, PDGF-B, VEGF, FGFb, EGF, IGF and HGF) versus inhibit hair follicle growth
(TGFb1). As PRP injection results in 300% to 700% enrichment of platelets secreting large
quantities of GFs that can have contradictory roles in hair follicle differentiation, we analyzed
platelet expression of GFs in PRP samples prepared from patient volunteers by quantitative
PCR. Expression of TGFb1 was highest followed by PDGF-A and PDGF-B while low to un-
detectable transcript levels were noticed for IGF and HGF. Variability in platelet expression
between patients was highest for TGFb1, PDGF-A and PDGF-B and lowest for VEGF.
Consistently, we detected high TGFb1 levels in PRP compared to other growth factors and its
concentration relative to PDGF-BB in PRP varied highly between patients. Thus, balance
between platelet-secreted TGFb1 and other GFs in PRP that promote hair follicle differenti-
ation could determine patient response to PRP therapy.
879
IL-9 mediated human primary keratinocytes invasion is dependent on MLC
controlled contractility and independent of MMP activity
S Das1
, S Srinivasan1
, A Srivastava1
, S Kumar2
, G Das3
, S Das1
, A Gupta4
, C Nayak4
and
R Purwar1
1 IIT Bombay, Mumbai, India, 2 Department of Biosciences & Bioengineering,
Mumbai, India, 3 Department of Biosciences and Bioengineering, Indian Institute of Tech-
nology, Bombay, Mumbai, India and 4 Topiwala National Medical College & B. Y. L. Nair
Charitable Hospital, Mumbai, India
T-helper 9 cells are recently discovered IL-9 secreting CD4+T helper cells and their presence
is described in healthy and skin inflammatory diseases. However, it remains poorly examined
how IL-9 impacts the cellular responses in the skin during homeostasis and disease patho-
genesis. In this study, we examined the roles of IL-9 in regulating the human primary kera-
tinocytes (HPKs) biophysical properties such cellular morphology, invasion and migration
potential, which are critical for maintaining skin homeostasis in healthy and diseased in-
dividuals. IL-9 promoted the random HPKs motility in 2D space as demonstrated by wound
healing and 2-D motility assays. In contrast, IL-9 down-regulated the HPKs invasion when
cells were embedded in 3D collagen matrix. Surprisingly, IL-9 mediated inhibition of cell
invasion was independent of matrix-metalloproteinase (MMPs) as neither the RNA levels nor
MMP activity changed upon IL-9 stimulation of HPKs. Further, we investigated the effect of IL-
9 on HPKs stiffness and contractility by atomic force microscopy and de-adhesion assay
respectively. Interestingly, IL-9 reduced the HPKs stiffness and also increased the cell circu-
larity which corroborated with reduction in its contractility. Finally, pharmacological inhi-
bition of MLC kinases demonstrated that IL-9 mediated loss of contractility and invasion
potential are dependent on Rho associated kinase (ROCK) and independent of MMP medi-
ated pathways. In conclusion, we show a novel mechanism by which IL-9 controls the HPKs
migration and invasion potential.
Growth Factors, Cell Adhesion and Matrix Biology | ABSTRACTS
www.jidonline.org S149