Regulation of Kv1 subunit expression in oligodendrocyte progenitor cells and their role in G1/S phase progression of the cell cycle

Abstract

Proliferative oligodendrocyte progenitor cells (OPs) express large, delayed outward-rectifying K(+) currents (I(K)), whereas nondividing immature and mature oligodendrocytes display much smaller I(K). Here, we show that up-regulation of I(K) occurs in G(1) phase of the cell cycle in purified cultured OPs and is the result of an RNA synthesis-dependent, selective increase of the K(+) channel subunit proteins Kv1.3 and Kv1.5. In oligodendrocyte cells acutely isolated from developing rat brain, a decrease of cyclin D expression is observed as these cells mature along their lineage. This is accompanied by a decrease in Kv1.3 and Kv1.5 subunit expression, suggesting a role for these subunits in the proliferative potential of OPs in situ. I(K) expressed in OPs in subventricular zone and developing white matter in acutely isolated slice preparations were selectively blocked by antagonists of Kv1.3, illustrating the functional presence of this subunit in situ. Interestingly, Kv1.3 block inhibited S-phase entry of both purified OPs in culture and in tissue slice cultures. Thus, we employ both in vitro and in situ experimental approaches to show that (i) RNA-dependent synthesis of Kv1.3 and Kv1.5 subunit proteins occurs in G(1) phase of the OP cell cycle and is responsible for the observed increase in I(K), and (ii) currents through Kv1.3-containing channels play a crucial role in G(1)/S transition of proliferating OPs.

Figure 1

I_K is up-regulated following 24-h (B) PDGF and (C) bFGF treatment (10 ng/ml), when compared with I_K in OPs in G₀ (DME-N1 medium) (A). (D and E) Time course of up-regulation of I_K in OPs following PDGF and bFGF (total cells = 131 and 147, respectively) treatment. (F and G) The PDGF- and bFGF-induced increase is not prevented by the G₁/S transition blockers, rapamycin (30 nM) and aphidicolin (3 μM), after 12 or 24 h following mitogen treatment (data represent means of 8–11 OP cell recordings per condition from at least three different individual cultures ± SEM). In F and G, I_k density in OP cells cultured in N1 medium without PDGF or bFGF was the same as in D and E.

Figure 2

Western blot analysis of Kv1.3–Kv1.6 expression in cultured OPs in DME-N1 medium and 24 h after PDGF (P) or bFGF (bF) treatment (10 ng/ml). A selective and significant increase of normalized protein expression (**, P < 0.01) is apparent following either PDGF or bFGF treatment of Kv1.3 (A) and Kv1.5 (C) but not of Kv1.4 (B) and Kv1.6 (D) (see Insets for single examples of band staining). Data were normalized with respect to cells cultured in DME-N1 and represent means from three to seven individual cultures ± SEM. Equal protein loading was verified by Ponceau S solution reversible staining of the blots.

Figure 3

(A and B) Densitometric measurement of Western blot band (see Insets) intensity illustrates that Kv1.3 and Kv1.5 protein expression is significantly prevented (*P < 0.05) by α-amanitin (+A; 20 μg/ml) in the presence of PDGF (P) and bFGF (bF) to levels that are comparable with those seen in OPs in G₀ (i.e., cultured in DME-N1). Data were normalized with respect to cells cultured in DME-N1 and represent means from three to five individual cultures ± SEM. (C and D) α-Amanitin also significantly prevents (*, P < 0.05) the mitogen-induced up-regulation (12- or 24-h treatment) of I_K density in OPs. Data represent the means of the calculated I_K density elicited by a test pulse to 50 mV and are from 15–21 OPs from at least three individual cultures. (E) Inhibition of RNA synthesis does not prevent the PDGF-induced entry of cultured OPs into the cell cycle, as assessed by cyclin D expression. Samples were processed for Western blot analysis with anti-cyclin D antibody at varying time points following PDGF treatment. ctr, purified cyclin D protein. Equal protein loading was verified by Ponceau S solution reversible staining of the blots.

Figure 4

(A) Acutely isolated cells were immunostained with oligodendrocyte lineage antibodies and anti-Kv1.3 or anti-Kv1.5 antibodies. A significant decrease in Kv1.3 and Kv1.5 expression was observed in O4⁺ and O1⁺ cells, as compared with NG2⁺ cells (**, P < 0.01; ***, P < 0.001). Data shown are means ± SEM obtained from three independent experiments. The number of total cells counted for each antibody ranged between 226 and 700. (B) Acutely isolated cells were immunostained with NG2, O4, O1, and anti-cyclin D antibodies. A significant decrease in cyclin D expression was observed in O4⁺ and O1⁺ cells, as compared with NG2⁺ cells (***, P < 0.001). The data shown are means ± SEM obtained from of three independent experiments. The total number of cells counted ranged between 456 and 1,042.

Figure 5

(A) A representative example of a biocytin-filled cell from which electrophysiological recordings were made. (B) Illustration that this cell (see arrow) was immunoreactive for NG2 (scale bars = 20 μM). (C) All cells expressed I_K (Middle) and, in some cases, I_A (64% of cells; n = 25, Bottom). Insets in top and middle panels illustrate voltage protocols used. (D) Activation curves for I_K illustrate the presence of two current components; data yielded a significantly better fit when described by the sum of two Boltzmann distributions (single Boltzmann vs. double Boltzmann fit; χ² values = 0.0013 ± 0.0001 vs. 0.0006 ± 0.00007; P < 0.001; n = 16). Data for the activation plots are expressed as means ± SEM from 25 cells. I_K was partially inhibited by either AgTx (50 nM) (E) or MgTx (50 nM) (F). Subsequent addition of TEA (10 mM) caused a further inhibition. Data are means ± SEM obtained from five to six cells. The traces in E and F are individual examples of I_K taken at the times indicated by the numbers.

Figure 6

(A) Cortical coronal slices were cultured for 48 h in the absence or presence of TEA (10 mM), r-agitoxin (AgTx; 50 nM), or r-margatoxin (MgTx; 50 nM) and incubated with BrdUrd for the last 24 h. Acutely isolated cell suspensions from these slices were immunostained with NG2 or O4 antibodies, together with anti-BrdUrd antibodies. All K⁺ channel blockers significantly inhibited cell proliferation in the NG2⁺ and O4⁺ cell populations (**, P < 0.01; ***, P < 0.001). Data shown are means ± SEM obtained from three to four experiments. Total number of NG2⁺ and O4⁺ cells counted for the different treatments ranged between 654 and 958. (B) Treatment of cultured OPs with AgTx or MgTx inhibits cell proliferation to the same extent as TEA. Data are means ± SEM of four to five independent experiments run in duplicate. The total number of cells counted for each treatment ranged between 2,843 and 3,616. (C) Time course of cyclin D expression following PDGF treatment of OPs in the absence or presence of r-agitoxin (AgTx; 5 and 50 nM) or r-margatoxin (MgTx; 5 and 50 nM).