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Nitric oxide synthase mediates PC12 differentiation induced by the surface topography of nanostructured TiO2.

Tamplenizza M, Lenardi C, Maffioli E, Nonnis S, Negri A, Forti S, Sogne E, De Astis S, Matteoli M, Schulte C, Milani P, Tedeschi G - J Nanobiotechnology (2013)

Bottom Line: Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF.We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2.Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: CIMAINA and Dipartimento di Fisica, Università degli Studi di Milano, via Celoria 16, Milano 20133, Italy. paolo.milani@mi.infn.it.

ABSTRACT

Background: Substrate nanoscale topography influences cell proliferation and differentiation through mechanisms that are at present poorly understood. In particular the molecular mechanism through which cells 'sense' and adapt to the substrate and activate specific intracellular signals, influencing cells survival and behavior, remains to be clarified.

Results: To characterize these processes at the molecular level we studied the differentiation of PC12 cells on nanostructured TiO2 films obtained by supersonic cluster beam deposition.Our findings indicate that, in PC12 cells grown without Nerve Growth Factor (NGF), the roughness of nanostructured TiO2 triggers neuritogenesis by activating the expression of nitric oxide synthase (NOS) and the phospho-extracellular signal-regulated kinase 1/2 (pERK1/2) signaling. Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF. We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2.

Conclusion: Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

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Related in: MedlinePlus

Involvement of ERK signaling cascade in nanostructured-induced neuritogenesis. The expression and the phosphorylation of ERK were evaluated by Western blot analysis using anti-ERK and anti-p-ERK antibodies, on PC12 cells grown on PLL-coated glass (G-PLL), flat Titania (flat TiO2) and nanostructured Titania (20 nm rms TiO2) in the presence and in the absence of 50 ng/mL NGF. Equal amounts of lysates (70 μg) were loaded on SDS-PAGE and probed with antibodies as indicated in the Methods section. The results are means of 3 different experiments.
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Figure 8: Involvement of ERK signaling cascade in nanostructured-induced neuritogenesis. The expression and the phosphorylation of ERK were evaluated by Western blot analysis using anti-ERK and anti-p-ERK antibodies, on PC12 cells grown on PLL-coated glass (G-PLL), flat Titania (flat TiO2) and nanostructured Titania (20 nm rms TiO2) in the presence and in the absence of 50 ng/mL NGF. Equal amounts of lysates (70 μg) were loaded on SDS-PAGE and probed with antibodies as indicated in the Methods section. The results are means of 3 different experiments.

Mentions: The addition of NGF to PC12 cells causes neurite elongation through a sustained activation of ERK, a mitogen-activated protein kinase whose phosphorylation is essential to neuronal differentiation [66]. As reported by Yamazaki et al. [30], this activation occurs upon activation of NOS and can be obtained also by NO itself, in the absence of NGF, during NO-induced neuritogenesis. These observations prompted us to check if the ERK-signaling cascade may be also involved in the differentiation process triggered by nanotopography. We checked the expression of ERK and its phosphorylation by Western blot analysis using anti-ERK and anti-p-ERK antibodies. The results, summarized in Figure 8, clearly show that when cells are grown on ns-TiO2 in NGF-free media ERK is phosphorylated to the same extent as in cell grown on glass or on flat TiO2 upon stimulation by NGF. In the latter two substrates the activation of ERK is almost undetectable in the absence of NGF.


Nitric oxide synthase mediates PC12 differentiation induced by the surface topography of nanostructured TiO2.

Tamplenizza M, Lenardi C, Maffioli E, Nonnis S, Negri A, Forti S, Sogne E, De Astis S, Matteoli M, Schulte C, Milani P, Tedeschi G - J Nanobiotechnology (2013)

Involvement of ERK signaling cascade in nanostructured-induced neuritogenesis. The expression and the phosphorylation of ERK were evaluated by Western blot analysis using anti-ERK and anti-p-ERK antibodies, on PC12 cells grown on PLL-coated glass (G-PLL), flat Titania (flat TiO2) and nanostructured Titania (20 nm rms TiO2) in the presence and in the absence of 50 ng/mL NGF. Equal amounts of lysates (70 μg) were loaded on SDS-PAGE and probed with antibodies as indicated in the Methods section. The results are means of 3 different experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3815074&req=5

Figure 8: Involvement of ERK signaling cascade in nanostructured-induced neuritogenesis. The expression and the phosphorylation of ERK were evaluated by Western blot analysis using anti-ERK and anti-p-ERK antibodies, on PC12 cells grown on PLL-coated glass (G-PLL), flat Titania (flat TiO2) and nanostructured Titania (20 nm rms TiO2) in the presence and in the absence of 50 ng/mL NGF. Equal amounts of lysates (70 μg) were loaded on SDS-PAGE and probed with antibodies as indicated in the Methods section. The results are means of 3 different experiments.
Mentions: The addition of NGF to PC12 cells causes neurite elongation through a sustained activation of ERK, a mitogen-activated protein kinase whose phosphorylation is essential to neuronal differentiation [66]. As reported by Yamazaki et al. [30], this activation occurs upon activation of NOS and can be obtained also by NO itself, in the absence of NGF, during NO-induced neuritogenesis. These observations prompted us to check if the ERK-signaling cascade may be also involved in the differentiation process triggered by nanotopography. We checked the expression of ERK and its phosphorylation by Western blot analysis using anti-ERK and anti-p-ERK antibodies. The results, summarized in Figure 8, clearly show that when cells are grown on ns-TiO2 in NGF-free media ERK is phosphorylated to the same extent as in cell grown on glass or on flat TiO2 upon stimulation by NGF. In the latter two substrates the activation of ERK is almost undetectable in the absence of NGF.

Bottom Line: Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF.We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2.Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: CIMAINA and Dipartimento di Fisica, Università degli Studi di Milano, via Celoria 16, Milano 20133, Italy. paolo.milani@mi.infn.it.

ABSTRACT

Background: Substrate nanoscale topography influences cell proliferation and differentiation through mechanisms that are at present poorly understood. In particular the molecular mechanism through which cells 'sense' and adapt to the substrate and activate specific intracellular signals, influencing cells survival and behavior, remains to be clarified.

Results: To characterize these processes at the molecular level we studied the differentiation of PC12 cells on nanostructured TiO2 films obtained by supersonic cluster beam deposition.Our findings indicate that, in PC12 cells grown without Nerve Growth Factor (NGF), the roughness of nanostructured TiO2 triggers neuritogenesis by activating the expression of nitric oxide synthase (NOS) and the phospho-extracellular signal-regulated kinase 1/2 (pERK1/2) signaling. Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF. We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2.

Conclusion: Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

Show MeSH
Related in: MedlinePlus