Pivotal Role of AKAP12 in the Regulation of Cellular Adhesion Dynamics: Control of Cytoskeletal Architecture, Cell Migration, and Mitogenic Signaling

Cellular dynamics are controlled by key signaling molecules such as cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). AKAP12/SSeCKS/Gravin (AKAP12) is a scaffold protein for PKA and PKC which controls actin-cytoskeleton reorganization in a spatiotemporal manner. AKAP12 also acts as a tumor suppressor which regulates cell-cycle progression and inhibits Src-mediated oncogenic signaling and cytoskeletal pathways. Reexpression of AKAP12 causes cell flattening, reorganization of the actin cytoskeleton, and the production of normalized focal adhesion structures. Downregulation of AKAP12 induces the formation of thickened, longitudinal stress fibers and the proliferation of adhesion complexes. AKAP12-null mouse embryonic fibroblasts exhibit hyperactivation of PKC, premature cellular senescence, and defects in cytokinesis, relating to the loss of PKC scaffolding activity by AKAP12. AKAP12-null mice exhibit increased cell senescence and increased susceptibility to carcinogen-induced oncogenesis. The paper describes the regulatory and scaffolding functions of AKAP12 and how it regulates cell adhesion, signaling, and oncogenic suppression

Production of H₂S is downregulated in replicatively senescent cells.

<p>(<b>A</b>) Representative images of SA-β-Gal staining in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. Scale bars, 100 μm. (<b>B</b>) Real-time PCR analysis of expression of <i>hTERT</i> in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. The expression of <i>hTERT</i> was normalized to the expression level of <i>β-ACTIN</i>. (<b>C</b>) NAD/NADH ratio in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. Real-time PCR analysis of expression of <i>CBS</i> (<b>D</b>), <i>MST</i> (<b>E</b>), and <i>CSE</i> (<b>F</b>) in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. The expression of <i>CBS</i>, <i>MST</i>, and <i>CSE</i> was normalized to the expression level of <i>β-ACTIN</i>. (<b>G</b>) 1 x 10⁶ cells of young (PD: 5.9) and senescent (PD: 18.8) aHDF cells were incubated in PBS at 37°C for 1 hour and then H₂S was measured in culture supernatants. Mean values are shown along with error bars. *; <i>p</i><0.05, **; <i>p</i><0.005, ***; <i>p</i><0.0005, n.s.; not significant.</p

NaHS-treatment increases expression of <i>NAMPT</i> and <i>SIRT1</i>.

<p>(<b>A</b> and <b>B</b>) The expression of <i>NAMPT</i> and <i>SIRT1</i> in young (PD: 5.9) and senescent (PD: 18.8) was assessed by real-time PCR and normalized to the expression level of <i>β-ACTIN</i>. (<b>C</b>) Immunoblotting of Nampt and Sirt1 in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. β-Actin was used as a loading control. (<b>D</b> and <b>E</b>) Young (PD: 5.9) aHDF cells were treated without and with NaHS for 3 days, and RNA samples were then subjected to real-time PCR for assessment of <i>NAMPT</i> and <i>SIRT1</i>. The expression levels of <i>NAMPT</i> and <i>SIRT1</i> were normalized to the levels of expression of <i>β-ACTIN</i>. (<b>F</b>) Immunoblotting of Nampt and Sirt1 in NaHS-treated young (PD: 5.9) aHDF cells. β-Actin was used as a loading control. (<b>G</b>) NAD/NADH ratio in young (PD: 5.9) aHDF cells treated without and with NaHS for 7 days. Data were normalized to the total amount of protein.</p

Exogenous H₂S increases PD and suppresses SA-β-Gal expression.

<p>(<b>A</b>) PD of cells treated without or with 1 μM NaHS. The population doubling of the first confluent cultures was designated as 0. (<b>B</b>) Representative images of SA–β-Gal staining in cells shown in Fig 3A. Mean values ± error bars of number of SA-β-Gal positive cells are shown on the right-upper corner of each image. *; p<0.05, ***; p<0.0005, n.s.; not significant. Scale bars, 100 μm.</p

Exogenous H₂S increases the expression of <i>hTERT</i> as well as the activity of telomerase.

<p>(<b>A</b>) Real-time PCR analysis of the expression of <i>hTERT</i> in young (PD: 5.9) aHDF cells, treated with NaHS for 3 days. The expression of <i>hTERT</i> was normalized to the level of expression of <i>β-ACTIN</i>. Expression of untreated control was regarded as 1.0. (<b>B</b>) Immunoblotting of hTERT in aHDF cells without or with 1 μM NaHS for 7 days. 100 μg of the indicated nuclear extracts were subjected for immunoblotting. β-Actin was used as a loading control. (<b>C</b>) Telomerase activity in young (PD: 3.2) aHDF cells without or with treated with 1 μM NaHS for 7 days. Positive control was MDA-MB-231 cell lysate, and negative control was buffer alone. Bottom panel shows quantified means ± error bars from three independent assays. Relative activity of telomerase was calculated by dividing the density of all ladders to the density of the bands in internal control, indicated as internal control (I.C.).</p

H₂S induces <i>hTERT</i> expression in a NAMPT/SIRT1-dependent manner.

<p>(<b>A</b> and <b>B</b>) Downregulation of <i>SIRT1</i> suppresses the expression of <i>hTERT</i>. Young (PD: 5.9) aHDF cells (3 x 10⁵ cells) were transfected with <i>SIRT1</i> siRNA for 2 days, and these were treated without or with NaHS for 3 days. Total RNAs from these cells were subjected to real-time PCR analysis for <i>SIRT1</i> (<b>A</b>) and <i>hTERT</i> (<b>B</b>). Data were normalized to the level of expression of <i>β-ACTIN</i>. The expression level of <i>SIRT1</i> and <i>hTERT</i> in cells treated with Scrambled siRNA without NaHS treatment was regarded as 1.0. (<b>C</b>) Downregulation of <i>NAMPT</i> suppresses the activity of SIRT1. Young (PD: 5.9) aHDF cells (3 x 10⁵ cells) were transfected with <i>NAMPT</i> siRNA for 2 days, and then the cells were treated without or with 1 μM NaHS for 3 days. Nuclear proteins were extracted and used for measurement of SIRT1 activity. Mean values ± error bars were normalized to the amount of total cell protein. (<b>D</b>) Mode of action of H₂S in opposing senescence. *; <i>p</i><0.05, **; <i>p</i><0.005, ***; <i>p</i><0.0005, n.s.; not significant.</p

Hydrogen sulfide facilitates reprogramming and trans-differentiation in 3D dermal fibroblast.

The efficiency of cell reprogramming in two-dimensional (2D) cultures is limited. Given that cellular stemness is intimately related to microenvironmental changes, 3D cell cultures have the potential of overcoming this limited capacity by allowing cells to self-organize by aggregation. In 3D space, cells interact more efficiently, modify their cellular topology, gene expression, signaling, and metabolism. It is yet not clear as how 3D culture environments modify the reprogramming potential of fibroblasts. We demonstrate that 3D spheroids from dermal fibroblasts formed under ultra-low attachment conditions showed increased lactate production. This is a requisite for cell reprogramming, increase their expression of pluripotency genes, such as OCT4, NANOG and SOX2, and display upregulated cystathionine-β-synthase (CBS) and hydrogen sulfide (H2S) production. Knockdown of CBS by RNAi suppresses lactic acid and H2S production and concomitantly decreases the expression of OCT4 and NANOG. On the contrary, H2S donors, NaHS and garlic-derived diallyl trisulfide (DATS), promote the expression of OCT4, and support osteogenic trans-differentiation of fibroblasts. These results demonstrate that CBS mediated release of H2S regulates the reprogramming of dermal fibroblasts grown in 3D cultures and supports their trans-differentiation

A H₂S-Nampt Dependent Energetic Circuit Is Critical to Survival and Cytoprotection from Damage in Cancer Cells

<div><p>We recently demonstrated that cancer cells that recover from damage exhibit increased aerobic glycolysis, however, the molecular mechanism by which cancer cells survive the damage and show increased aerobic glycolysis remains unknown. Here, we demonstrate that diverse cancer cells that survive hypoxic or oxidative damage show rapid cell proliferation, and develop tolerance to damage associated with increased production of hydrogen sulfide (H₂S) which drives up-regulation of nicotinamide phosphoribosyltransferase (Nampt). Consistent with existence of a H₂S-Nampt energetic circuit, in damage recovered cancer cells, H₂S, Nampt and ATP production exhibit a significant correlation. Moreover, the treatment of cancer cells with H₂S donor, NaHS, coordinately increases Nampt and ATP levels, and protects cells from drug induced damage. Inhibition of cystathionine beta synthase (CBS) or cystathionase (CTH), enzymes which drive generation of H₂S, decreases Nampt production while suppression of Nampt pathway by FK866, decreases H₂S and ATP levels. Damage recovered cells isolated from tumors grown subcutaneously in athymic mice also show increased production of H₂S, Nampt and ATP levels, associated with increased glycolysis and rapid proliferation. Together, these data show that upon recovery from potential lethal damage, H₂S-Nampt directs energy expenditure and aerobic glycolysis in cancer cells, leads to their exponential growth, and causes a high degree of tolerance to damage. Identification of H₂S-Nampt as a pathway responsible for induction of damage tolerance in cancer cells may underlie resistance to therapy and offers the opportunity to target this pathway as a means in treatment of cancer.</p></div

Damage-Recovered (DR) cells show increase in H₂S and proliferation rate and exhibit tolerance to damage.

<p>(A) A scheme for isolation of Damage-Recovered (DR) cells. (B) Bax expression in H₂O₂ treated Pc, DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells. (C) Amount of H₂S released by DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells. Significance between Pc and three DR cells was <i>p</i><0.0005 in ANOVA statistical analysis. (D) H₂S staining of Pc, DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells with 5 µM H₂S fluorescent probe, HSN2. Scale bars, 50 µm. (E) PCR analysis of <i>CBS</i>, <i>CTH</i> and <i>MTS</i> genes in Pc, DR^{H2O2 W1} and DR^{H2O2 W3} HepG2 cells. (F) Western blot analysis of CBS and CTH in Pc, DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells. (G) Western blot analysis of CBS in Pc and DR^{H2O2 W1}, DR^{H W1} and DR^{G W1} HepG2 cells. (H) Proliferation of HepG2 recovered from H₂O₂, DR^{H2O2 W1}, DR^{H2O2 W2} cells as a percentage of that in Pc cells. (I) Viability of Pc, DR^{H2O2 W1} and DR^{H2O2 W2} HepG2 cells with and without treatment with bleomycin. *; <i>p</i><0.05,**; <i>p</i><0.005, ***; <i>p</i><0.0005.</p