The goal of this study was to determine whether the presence of different E-peptides affected the production, distribution, or stability of the mature IGF-I protein. In addition to expressing IGF-IA and IGF-IB, a series of expression constructs based on the IGF-IA and IGF-IB open reading frames were generated to enable the expression of mature IGF-I in the absence of either EA- or EB-peptides (IGF-IStop) or the expression of either EA- or EB-peptide in the absence of mature IGF-I (SigEA, and SigEB, respectively). In addition, cleavage mutants were generated with the intent of expressing only proIGF-IA and proIGF-IB (IGF-IA.K68G and IGF-IB.K68G, respectively), and inhibiting the ability of mature-IGF-I production.
Transfection and Expression Efficiency
The efficiency of transfection was determined for each construct by the proportion of GFP-positive cells in each dish and by qRT-PCR. The proportion of GFP-positive cells did not differ among the constructs, resulting in a combined transfection efficiency of 12.7 ± 1% (mean ± SD). Transfection efficiency also was determined by measuring the level of GFP expression with respect to a housekeeping gene (18s) and by comparing GFP expression by each plasmid to the empty vector control. As shown in A, there was no significant difference in the relative expression of GFP between any of the transfected constructs.
Figure 2. Transfection and expression efficiency for IGF-I constructs. Data are presented as mean and SEs from three independent experiments. (A) Efficiency of transfection was determined in using the relative GFP expression compared with 18s as a housekeeping (more ...)
Validation of expression was achieved by qRT-PCR for each construct by using primers specific to each IGF-I insert. All transfections expressed the insert of interest >3000-fold higher than in controls. The efficiency of expression in each transfection experiment was determined by comparing the calculated transcript copies for each IGF-I cDNA insert to the calculated transcript copies for GFP. As shown in B, there was no significant difference in expression efficiency between constructs. Therefore, similar efficiencies of both transfection and expression were observed for all IGF-I constructs.
Secretion of IGF-I
Secretion of IGF-I from cells into the media was measured by ELISA 24 h after transfection (). IGF-I secretion was significantly higher from transfected cells than controls when the transfection construct retained the mature IGF-I protein coding sequence. Secretion was not affected by the presence or absence of EA or EB, for IGF-I secretion from cells transfected with the IGF-IA, IGF-IB, IGF-IStop constructs was equivalent. Secretion of IGF-I from cells transfected with the cleavage mutant constructs (IGF-IA.K68G and IGF-IB.K68G) did not differ from IGF-IA or IGF-IB. However, there was more IGF-I secreted from IGF-IB.K68G transfections than from cells transfected with IGF-IA.K68G. Endogenous IGF-I secretion was not altered by transfection agent (mock), the transfection vector (GFP), or the transfection of SigEA or SigEB constructs. Levels of IGF-I produced by these cultures were between 4 and 10 pg/ml and were not visible in the scale in . Secretion of IGF-I also was determined in cell transfections of epitope-tagged constructs. The presence of FLAG or HA on the constructs did not affect the secretion of IGF-I from the cells (data not shown).
Figure 3. IGF-I production is not affected by the presence of the E-peptides. Media content of IGF-I served as an index of IGF-I production after transfection. Production of IGF-I was significantly higher than control cells when the cDNA construct contained the (more ...)
Processing of IGF-I from proIGF-I to mature IGF-I can occur both intracellularly and extracellularly in a number of cell types (Conover et al., 1989
; Duguay et al., 1997
; Duguay, 1999
; Wilson et al., 2001
). To determine the forms of IGF-I that were secreted from transfected C2C12 cells, the media from FLAG-IGF-IA, FLAG-IGF-IB, and FLAG-IGF-IStop transfected cells was subjected to immunoblotting to detect FLAG-labeled IGF-I. When the E-peptides were present in the construct, both proIGF-I and mature IGF-I could be detected in the media (). FLAG-IGF-IA transfected cultures had an additional higher molecular weight band (Gly-ProIGF-I) consistent with glycosylation of the EA-peptide (Bach et al., 1990
; Duguay et al., 1995
; Wilson et al., 2001
). In FLAG-IGF-IB–transfected cultures, an additional band that was smaller than proIGF-I was evident (band c, ProIGF-I'). In FLAG-IGF-Istop transfected cultures, the mature IGF-I band was apparent, as well as one higher molecular weight band that was not evident in the IGF-IA and IGF-IB lanes.
Figure 4. Form of secreted IGF-I from C2C12 cells after transfection. Immunoblotting of concentrated media with anti-FLAG was used to distinguish between proIGF-I and mature IGF-I in the culture media (left). Both pro- (bands a and b) and fully processed (mature, (more ...)
Cellular Distribution of IGF-I and E-Peptides
Localization of IGF-I and the E-peptides was assessed by immunocytochemistry of the FLAG and HA epitope tags 24 h after transfection ( and ). All GFP-positive cells were positive for FLAG or for HA when these epitope tags were in the cDNA constructs. GFP was found in the cytoplasm and nucleus of positively transfected cells. FLAG and HA staining in the GFP-positive cells was concentrated in the perinuclear region but also could be detected throughout the cell. Expression of the cleavage mutant constructs (IGF-IAK68G and IGF-IBK68G) altered cell shape (, d and e, and , e and f). First, these cells had more cytoplasmic extensions than in other conditions for both FLAG- and HA-labeled constructs. Second, FLAG and HA staining in these cells seemed restricted to the perinuclear regions in contrast to the more widespread distribution found in cells transfected with the other constructs. FLAG-tagged IGF-IA, IGF-IB, and IGF-IStop had similar localization patterns in the GFP-positive cells (, a–c, respectively). HA-tagged IGF-IA, IGF-IB, SigEA, and SigEB also had similar staining patterns in the GFP-positive cells (, a–d). The pattern of HA staining was independent of the IGF isoform that had been transfected.
Figure 5. Cellular distribution of FLAG epitope-tagged IGF-I constructs FLAG-IGF-IA (a), FLAG-IGF-IB (b), FLAG-IGF-Istop (c), FLAG-IGF-IAK68G (d), FLAG-IGF-IBK68G (e), and no transfection (f). GFP serves as an indicator of positive transfection and is found in (more ...)
Figure 6. Cellular distribution of HA epitope-tagged IGF-I constructs HA-IGF-IA (a), HA-IGF-IB (b), HA-SigEA (c), HA-SigEB (d), HA-IGF-IAK68G (e), and HA-IGF-IBK68G (f). GFP serves as an indicator of positive transfection and is found in the cytoplasm and nucleus (more ...)
GFP-negative cells that were FLAG or HA positive served as indicators of internalization of IGF-I or E-peptide, respectively. We took advantage of the mixed population of transfected and nontransfected cells within each culture dish to evaluate cell entry of FLAG as an indicator of IGF-I entry and HA as an indicator of EA or EB entry. Within the GFP-negative cells of each transfection experiment, the percentage of FLAG- or HA-positive cells was quantified ().
Figure 7. Proportion of C2C12 cells that internalize epitope tags after transfection. (A) FLAG uptake is dependent upon the transfected IGF-I construct. There was no difference in the proportion of FLAG-positive cells after transfection of FLAG-IGF-IA or FLAG-IGF-IB. (more ...)
Most of the cells in FLAG-IGF-IA or FLAG-IGF-IB transfections were FLAG positive, and there was no statistical difference in FLAG (IGF-I) uptake between these isoforms (A). However, FLAG-positive cells in FLAG-IGF-IStop-transfected cultures were significantly lower than the constructs retaining the E-peptides (IGF-IA and IGF-IB). Therefore, presence of the E-peptides in the cDNA construct altered the proportion of FLAG-positive cells. Transfection of cells with the cleavage mutant construct FLAG-IGF-IAK68G resulted in a significant decrease in FLAG-positive cells compared with FLAG-IGF-IA. There was no statistical difference in the proportion of FLAG-positive cells between FLAG-IGF-IB and FLAG- IGF-IBK68G. Therefore, inhibition of the native cleavage site between mature IGF-I and the EA-peptide seemed to be important for normal uptake of IGF-I into neighboring cells. HA was detected in a lower proportion of nontransfected cells than FLAG (B). There were no statistical differences in the percentage of HA-positive cells among all of the HA epitope-tagged constructs. Therefore, E-peptide internalization seems independent of IGF-I.