Sensitization of Transforming Growth Factor‑β Signaling by Multiple Peptides Patterned on DNA Nanostructures

We report sensitization of a cellular signaling pathway by addition of functionalized DNA nanostructures. Signaling by transforming growth factor β (TGFβ) has been shown to be dependent on receptor clustering. By patterning a DNA nanostructure with closely spaced peptides that bind to TGFβ receptor, we observe increased sensitivity of NMuMG cells to TGFβ ligand. This is evidenced by translocation of secondary messenger proteins to the nucleus and stimulation of an inducible luciferase reporter at lower concentrations of TGFβ ligand. We believe this represents an important initial step toward realization of DNA as a self-assembling and biologically compatible material for use in tissue engineering and drug delivery

The effect of knockdown on hASC proliferation in expansion media (CGM).

<p>DNA was collected at days 0, 3, 7 and 10 to measure proliferative activity with siRNA knockdown of PC1, PC2 and IFT88 in as compared to the non-targeting scramble control siRNA. Knockdown of IFT88 significantly increases hASC proliferation as compared to control (n = 3, error bars = SEM, Student's t-test comparing each treatment to control, * p<0.05, ** p<0.01).</p

Primary cilia on hASC.

<p>(a) The non-motile primary cilium axoneme is composed of 9 pairs of peripheral microtubule doublets arranged concentrically in 9+0 configuration, devoid of a central tubulin pair. (b–f) Primary cilia appear as a dense cluster of acetylated tubulin protruding from the apical surface of hASC in confluent culture. (b) Few cilia are observed 24 hours after culture in complete growth expansion media (CGM) in subconfluent culture. They are observed at day 3 (c) undifferentiated in CGM, (d) differentiated in osteogenic differentiation medium (ODM) and at day 12 in (e) undifferentiated in CGM and (f) differentiated in ODM. Cilia are identified with anti-acetylated tubulin (green) or in combination with CEP164 (red), actin identified with phalloidin (red) and nuclei are identified with DAPI staining (blue). Scale bar = 25 µm.</p

Osteogenic gene marker expression in hASC.

<p>IFT88 knockdown confers a reduction in (a) Runx2 gene expression after 3 day and (c) BMP-2 gene expression after 10 day culture in osteogenic differentiation medium (ODM). (b) PC1, PC2 and IFT88 knockdown confer a reduction in alkaline phosphatase (ALP) gene expression after 10 days of culture in ODM. (d) PC1 knockdown confers downregulation of osteocalcin after 15 days of culture in osteogenic differentiation. Knockdown treatments applied at day 0 and day 7 of differentiation, (n = 3, error bars = SEM, Student's t-test comparing each treatment to control, * p<0.05, ** p<0.01).</p

Endogenous alkaline phosphatase (ALP) activity of hASC after 14 days of culture.

<p>hASC were cultured in osteogenic differentiation medium (ODM) and transfected with non-targeting scramble, PC1, PC2 and IFT88 siRNA. ODM and complete growth medium (CGM) hASC without knockdown (no KD) were also measured for comparison of baseline activity. Alkaline phosphatase activity is suppressed with cilia-associated protein knockdown. Knockdown of PC1 (*** p<0.01), PC2 (* p<0.05) and IFT88 (* p<0.05) all led to a significant decrease in ALP enzymatic activity compared to the non-targeting scramble siRNA knockdown control (n = 3, error bars = SEM, One-way repeated measures ANOVA, with Newman-Keuls post-hoc test).</p

PC1, PC2 and IFT88 gene expression up to 15 days in ODM culture.

<p>siRNA knockdown applied at days 0 and 7, confers at least a 50% reduction in mRNA expression of each cilia-associate protein (n = 3, Error bars = SEM).</p

Calcium accretion is affected by cilia-associated protein knockdown.

<p>(a) Alizarin Red staining indicating degree of mineralization after 14 days ODM with knockdown. (b) Quantified calcium accretion normalized to DNA content. PC1 and PC2 knockdown leads to diminished calcium accretion, (n = 3, error bars = SEM, Student's t-test comparing each treatment to control, * p<0.05).</p