The Small Conductance Calcium-activated Potassium Channel Regulates Ion Channel Expression in C3H10T1/2 Cells Ectopically Expressing the Muscle Regulatory Factor MRF4

Abstract

We investigated small conductance (SK) potassium channel-mediated regulation of muscle-specific, ion channel functional expression in the C3H10T1/2-MRF4 cell model system, a stable fibroblast line ectopically overexpressing the myogenic regulatory transcription factor, MRF4. Mitogenic stimulation of C3H10T1/2-MRF4 cells with basic fibroblast growth factor negatively regulates MRF4 transcriptional activity, inhibiting myogenesis. Using patch clamp techniques we found that mitogenic stimulation of C3H10T1/2-MRF4 cells also up-regulated SK. SK is a charybdotoxin-sensitive, apamin-insensitive channel that exerts positive proliferative control in fibroblasts. Mitogen withdrawal, which removes negative regulation of MRF4 thus initiating myogenesis, also eliminated SK channel currents, coincident both with induction of acetylcholine receptor channels, and up-regulation of muscle inward rectifier potassium channels. Addition of the SK channel blocker charybdotoxin to growth factor-containing culture medium overcame basic fibroblast growth factorinduced negative regulation of MRF4, as evidenced by induction of inward rectifier potassium and acetylcholine receptor channel expression identical to that observed in mitogen-withdrawn cells. Thus, the SK channel can govern electrophysiological phenotype in C3H10T1/2-MRF4 cells, consistent with an ability of SK to affect MRF4-dependent transcriptional activity. SK appears to be a pivotal signaling component for growth factor regulation of both cell proliferation and differentiation.The regulated expression of ion channels is a key component of developmental processes in many cell types. In nonexcitable cells such as lymphocytes and fibroblasts, both potassium and voltage-independent cation channels contribute to proliferative and cell fate control mechanisms (1-6), while in excitable tissues, the expression of both voltage-dependent cation and ligand gated channels is a culminating event in cell differentiation. The latter scenario is prominent in mammalian skeletal muscle, for which cell maturation is marked by the up-regulation of voltage-dependent channels and conditional expression of different ACh 1 receptor channel subtypes (7). In turn, the progression of muscle fiber differentiation may be affected by the activity of these channels (8 -10).To understand ion channels as both causal and effector agents in cell growth and differentiation, we have looked at the regulation of channel functional expression in the C3H10T1/2-MRF4 myogenic model cell line. C3H10T1/2 (10T1/2) is a multipotent, fibroblast-like cell line that can be manipulated to express a phenotype characteristic of either chondrocytes, adipocytes, or myocytes (11). A muscle phenotype can be selectively produced in 10T1/2 cells via overexpression of the myogenic regulatory transcription factor MRF4. In the presence of bFGF or other mitogenic stimuli, MRF4 is unable to initiate muscle-specific gene expression, even though MRF4 protein levels remain unchanged. Upon bFGF withdrawal, negative regulation of MRF4 is re...

Ras/MEK/ERK Up-regulation of the Fibroblast KCaChannel FIK Is a Common Mechanism for Basic Fibroblast Growth Factor and Transforming Growth Factor-β Suppression of Myogenesis

“…EGF activation of Ras/MEK/ERK produces only transient FIK up-regulation in 10T1/2-MRF4 (and other fibroblast) cells (6), consistent with its poor myogenic suppressive action. We show here that the myogenic inhibitory action of Ras/MEK/ERK-induced FIK up-regulation is restricted to the suppression of only certain muscle-specific genes such as the ACh receptor channel, the expression of which we have shown precedes other myogenic events such as myosin expression and myotube formation (4). The selective inhibition of early versus late myogenic events may indicate a critical role for FIK in the physiological control of myogenesis by bFGF and TGF-␤.…”

“…10T1/2-MRF4 cultures were grown on gelatin-coated dishes as undifferentiated myoblasts in serum-replete medium (basal medium Eagle, 15% fetal bovine serum), or they were differentiated via serum withdrawal as described previously (4). For electrophysiology cells were seeded onto 35-mm dishes and grown to confluence in serum-replete medium and then switched to low glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 2% horse serum (HS).…”

“…Electrophysiology-Patch clamp apparatus, techniques, and cell preparation were as described previously (4,5). Whole-cell FIK currents were measured during voltage steps to 0 mV (from Ϫ70mV) while intracellular calcium levels were increased by extracellular application of A23187 (1 M).…”

“…Pathway-bFGF upregulates FIK in 10T1/2-MRF4 cells (4), and increased FIK channel activity stimulates proliferation and suppresses myogenesis. To date, additional growth factor-activated signaling systems that modulate FIK, and thus may regulate muscle cell growth and differentiation, have not been identified.…”

“…FIK in turn modulates and specifically controls proliferation and differentiation in this and other cell types (4,5). Thus FIK may be a critical component of growth factor signaling pathways that regulate proliferation and differentiation in skeletal muscle myoblasts.…”