Regulating the availability of transforming growth factor ß1 in B104 neuroblastoma cells

Abstract

Transforming growth factor (TGF) beta1 is a key player in early brain development, hence, its availability (i.e., synthesis and release) affects neuronogenesis. TGFbeta1 moves proliferating cells out of the cell cycle and promotes their subsequent migration. The present study tested the hypothesis that neural progenitors self-regulate TGFbeta1. B104 neuroblastoma cells which can grow in the absence of serum or growth factors were used in systematic studies of transcription, translation, release, and activation. These studies relied on quantitative enzyme-linked immunosorbent assays and real-time polymerase chain reactions. TGFbeta1 positively upregulated its own intracellular expression and promoted increased release of TGFbeta1 from cells. The induction of TGFbeta1 was independent of a change in transcription, but it depended on cycloheximide-inhibited translation. Signaling mediated by downstream Smad2/3 through the TGFbeta receptors and intracellular protein transport were also required for release of TGFbeta1 from B104 cells. Thus, TGFbeta1 production and release were mediated through a feed-forward mechanism and were pivotally regulated at the level of translation. These activities appear to be key for the role of TGFbeta1 in the proliferation and migration of young neurons.

Figure 1. Effects of TGFβ1 on the numbers of B104 cells

The number of cells in untreated cultures (solid bars) increased over time. Treatment with TGFβ1 (1.0 ng/ml; open bars) impeded this increase. Each value is the mean of three to five samples (± the standard error of the mean). Statistical significance is denoted by *, #, and @ for differences relative to the controls at the same time, to samples at the same condition at 6 hr, and to samples at the same condition at 24 hr, respectively.

Figure 2. TGFβ1 content in the medium

The amount of TGFβ1 (total and active) was determined using quantitative ELISAs. Latent TGFβ1 content was calculated as the difference between the concentrations of total and active TGFβ1. Cells were raised in a serum-free medium (solid bars) or treated with the same medium supplemented with TGFβ1 (1.0 ng/ml; open bars). The upper set of graphs describes the concentration of TGFβ1 per ml medium, whereas the lower triad of graphs depicts the same data in terms of TGFβ1 concentration per million cells. The number of cells in the cultures varied over time and was affected by TGFβ1 treatment. Therefore, the concentration of TGFβ1 was calculated in relation to the number of cells in the sample as depicted in the lower graphs. Notations as in Figure 1. n = 4 to 6.

Figure 3. TGFβ1 degradation

The change in the concentration of TGFβ1 over time was traced in medium without cells. Each datum is the mean of four samples (± the standard error of the mean).

Figure 4. TGFβ1 content in the cell lysate

TGFβ1 content following TGFβ1 exposure (1.0 ng/ml) was determined by ELISA and expressed relative to the number of cells present. Notations as in Figure 1. n = 3 to 5.

Figure 5. TGFβ1 transcript expression

The effect of exogenous TGFβ1 (1.0 ng/ml) on the expression of its own transcript was determined using qualitative real-time polymerase chain reaction. Changes in TGFβ1 transcript were normalized to expression of 18S ribosomal RNA and expressed as in terms of fold change relative to the amount of transcript in 3 hr control. A best-fit line was generated by linear regression. n = 3 to 5.

Figure 6. Prior cycloheximde exposure inhibits TGFβ1-induction in cell lysates

The TGFβ1 content of cell lysates was quantified by ELISA and normalized to cell counts. One hour of exposure to vehicle or cycloheximide (10 ng/ml in DMSO) was followed by timed exposure to TGFβ1 (0 or 1.0 ng/ml) with continued vehicle or cycloheximide exposure. Statistically significant differences are identified by the following symbols: * for differences relative to the controls as the same time, % for differences relative to the samples treated with TGFβ1 at the same time, and # and @ for similarly treated samples at the 1 hr and 6 hr timepoints, respectively. n = 3 to 5.

Figure 7. Cycloheximide blocks TGFβ1-induced latent TGFβ1 release

Following 24 hr of exposure to TGFβ1 (0 or 1.0 ng/ml), B104 cells were treated for an additional 24 hr to serum-free medium (none), vehicle (DMSO), or cycloheximide (10 ng/ml). The TGFβ1 content of conditioned medium (top) and cell lysates (bottom) was quantified and normalized to cell counts. Each * denotes a statistically significant (p<0.05) difference relative to control for the same treatment. A # identifies a difference relative to cells raised in a serum-free condition. An @ signifies a difference relative to samples exposed to the vehicle DMSO. n = 3 to 6.

Figure 8. Intracellular transport inhibitors block TGFβ1 release

Cells were exposed to TGFβ1 (1.0 ng/ml) for 24 hr followed by a 6 hr exposure to protein transport inhibitors (GolgiStop and GolgiPlug) with or without cycloheximide (10 ng/ml). TGFβ1 content of the medium (top) and cell lysate (bottom) were determined by ELISA. An * denotes a statistically significant (p<0.05) difference relative to control for the same treatment. A # identifies a difference relative to samples exposed to the vehicle DMSO. An @ signifies a difference relative to GolgiStop or GolgiPlug for the same condition. A % designates a significant difference for the comparison of GolgiStop or GolgiPlug for the same condition. n = 3 to 6.

Figure 9. TGFβ1-induced TGFβ1 release requires TGFβr signaling through Smad2/3

TGFβ1 content of the medium (top) and cell lysate (bottom) were assayed following 48 hr of simultaneous exposure to TGFβ1 (0 or 1.0 ng/ml) and the Smad2/3 inhibitor SB431542 (0 or 10 μm). Notations as in Figure 6. n = 4 to 6.