Characterization of ET1ep-/- mouse line

By Steven MaMentor: Dr. Arup Indra

Pharmaceutical SciencesSep 25, 2009

Why ET1 (endothelin-1)?• Known growth factor –

hyperproliferative pathologies: psoriasis and cancer?

• Endothelin “axis” has been shown to promote cancer progression (Bagnato et al. 2008)

o Melanomao Breast, prostate, brain, lung, bladder etc.

• Malignant melanoma is the leading cause of death (75%) in skin cancers - no curative measures yet (Jerant et al. 2000)

EndothelinsWhat we know:• 21 amino acid

peptides• Three isoforms, ET-

1, ET-2, ET-3• Produced by

keratinocytes at a basal level

http://upload.wikimedia.org/wikipedia/commons/a/ac/1EDP_human_endothelin1_03.png

What we don’t know:• Autocrine functions in keratinocytes

Overview of Skin• Many Layers95% are keratinocytes

• Melanocyteso Secrete melanin – responsible for pigmentation (and tanning)

o Derived from neural crest cells (same as neurons)

Diferentiation

Proliferation

Hypothesis

ET1 has an autocrine effecton epidermal keratinocyte homeostasis, altering cellproliferation and differentiation.

ET1 has a paracrine effect on melanocytes.

Getting ET-1 out of epidermis

• Bacteriophage P1 site-specific Cre-lox recombination

• Flank target genes with loxP sites

• Cre – Cyclic Recombinase

• Human – mice homology http://upload.wikimedia.org/wikipedia/commons/5/58/CreLoxP_experiment.png

x

Techniques

• PCR: to confirm ET-1 ablation in epidermis

• Paraffin sections of skin treated with 4% PFA (paraformaldehyde) used for:

Hematoxylin and eosin staining

ImmunohistochemistryFontana Masson staining

Hematoxylin & eosin staining (HE)

• Stains nucleus blue and cytoplasm pink

• Popular method in histology, widely used in medical diagnosis, e.g. tumor biopsies

• Used to measure epidermal thickness

HFHFD

E{

DHF HF

E{

Data: epidermal thickness

ET1L2/L2 (wildtype)

ET1ep-/- (knockout)

ET1L2/L2 (wildtype) ET1ep-/- Ep

ider

mal

thick

ness

(µ

m)

No significance

Immunohistochemistry (IHC - with paraffin sections)

• Use antibodies to detect specific proteins in skin sections

• Purpose: analyze cell differentiation and proliferation

• Used Cy3 2ndary antibody

Epidermal Proteins

Loricrin

Keratin 10 (K10)Keratin 14(K14)

Late differentiating cells

Late differentiating cells

Early proliferating cells

Ki 67 Proliferation marker; present in all active cell phases (but not G0)

Red – Cy3Blue – Dapi (all cells)ET1L2/L2 (Wildtype) ET1ep-/-

Immunohistochemistry – differentiation

DE

K14

E

D

K10

D

E Loricrin

Red – Cy3Blue – Dapi (all cells)ET1L2/L2 (Wildtype) ET1ep-/-

Immunohistochemistry – proliferation

E

D

Ki 67

Counts (per 100 cells):

ET1L2/L2

(Wildtype)ET1ep-/-

(knockout)

7.68 5.57

Fontana-Masson Staining (FM)

Stains all nuclei red, but melanocytes black

• “Melanin stain”

• Used to analyze paracrine effect of keratinocyte ET-1 on neighboring melanocytes

Fontana-Masson StainET1L2/L2 (Wildtype) ET1ep-/-

Fontana-Masson Stain:melanocyte count compared

Results thus far• No significant

autocrine effect on keratinocytes:– Proliferation– differentiation

• Significant effect on melanocyte proliferation

• Adult mice

Ongoing experiments• UV treatment to new born

mice–Skin samples at 0h, 24h,

48h time points–Fontana-Masson staining

analysis• qPCR for melanogensis

factors in UV treated mice

Fontana-Masson Stain:UV treatment – 0 hour time point

Special thanks to:

Dr. Kevin Ahern Dr. Arup IndraSteven Hyter Dr. Gitali IndraXiaobo Liang Gaurav BajajDan Coleman Zhixing Wang

References• Jerant, A; Johnson, J; Sheridan, C; Cafferey, T.

“Early Detection and Treatment of Skin Cancer.” American Family Physician. July 15, 2000

• Bagnato, A; Spinella, F; Rosano, L. “The endothelin axis in cancer: the promise and the challenges of molecularly targeted therapy. Can J Physiol Pharmacol. August, 2008. p473-84

Questions?