Unexpected evolutionarily conserved rapid effects of viral infection on oxytocin receptor and TGF-β/pSmad3

Abstract

Background: shRNA lentiviral vectors are extensively used for gene knockdowns in mammalian cells, and non-target shRNAs typically are considered the proper experimental control for general changes caused by RNAi. However, the effects of non-target lentivirus controls on the modulation of cell signaling pathways remain largely unknown. In this study, we evaluated the effect of control lentiviral transduction on oxytocin receptor (OXTR) expression through the ERK/MAPK pathway in mouse and human skeletal muscle cells, on myogenic activity, and in vivo on mouse muscle regeneration. Furthermore, we mined published data for the influence of viral infections on OXTR levels in human populations and found that unrelated viral pathologies have a common consequence: diminished levels of OXTR.

Methods: We examined the change in OXTR mRNA expression upon transduction with control and Smad3-targeting viral vectors through real time RT-PCR and Western blotting, and confirmed with immunofluorescence. Changes in Smad3 and OXTR expression were examined both in vitro with mouse and human myoblasts and in vivo in mouse satellite cells. The general effects of viral infections on OXTR downregulation in humans were also examined by analyzing published Gene Expression Omnibus (GEO) datasets. The change in myoblast myogenic activity caused by the viral transduction (the percent of Pax7 + Ki67+ cells) was examined by immunofluorescence.

Results: Results shown in this work establish that lentiviral control vectors significantly downregulate OXTR expression at mRNA and protein levels and diminish key downstream effectors of OXTR, ERK signaling, reducing the myogenic proliferation of infected cells. This effect is evolutionarily conserved between mouse and human myogenic cells, and it manifests in satellite cells after control lentiviral transduction of mice in vivo. Furthermore, an examination of published datasets uncovered similar OXTR downregulation in humans that are afflicted with different viral infections. Additionally, cells transduced with Smad3-targeting shRNA downregulate OXTR even more than cells transduced with control viruses.

Conclusions: Our work suggests that experimental cohorts transduced with control viruses may not behave the same as un-transduced cells and animals, specifically that control viral vectors significantly change the intensity of key cell-signaling pathways, such as OXTR/ERK. Our results further demonstrate that lentiviral transduction significantly decreases myogenic proliferation and suggest that viral infections in general may play a role in decreasing muscle health and regeneration, a decline in metabolic health, and a lower sense of well-being, as these rely on effective OXTR signaling. Additionally, our data suggest pathway crosstalk between TGF-β/pSmad3 and OXTR, implying that sustained attenuation of the TGF-β/pSmad3 pathway will reduce pro-regenerative OXTR/pERK signaling.

Keywords: ERK; Muscle Regeneration; Oxytocin Receptor; RNAi; Smad; TGF-β; Viral Vectors; Viral infections.

Fig. 1

OXTR expression is downregulated upon lentiviral transduction. a. qRT-PCR in primary mouse myoblasts transduced in vitro with non-target (GFP) versus Smad3-targeting shRNA vectors at MOI = 0.5. OXTR expression is attenuated by all lentiviral vectors while Smad3 is attenuated only by Smad3-targeting shRNA vectors. a) Shows combined results from three shRNAs targeting Smad3. Results of individual shRNAs to Smad3 are shown in Figure 1SA. For mouse myoblasts: N = 6 for non-target shRNA, N = 8 for Smad3-targeting shRNA. b. OXTR is downregulated in muscle satellite cells in vivo with both non-target (GFP) shRNA and Smad3-targeting shRNA lentiviruses, but only Smad3 targeting diminished the Smad3 levels. Comparison between the level of OXTR in primary myoblasts and in vivo satellite cells is shown in Additional file 1: Figure S1B. *P ≤ 0.05, ** P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. For in vivo: * P ≤ 0.05; **P ≤ 0.01, ***P ≤ 0.001

Fig. 2

OXTR downregulation upon lentiviral transduction is evolutionarily conserved between mice and human. Shown are real-time qRT-PCR results on RNA derived from primary human myoblasts, which were transduced in vitro with non-target (GFP) versus Smad3-targeting shRNA vectors at MOI = 0.5. OXTR expression is attenuated by both lentiviral vectors while Smad3 is attenuated only by Smad3-targeting shRNA vectors. This downregulation of OXTR by lentiviral transduction is evolutionarily conserved between mouse and human

Fig. 3

Human database analysis reveals downregulation of OXTR expression upon different viral transfections. a. Analysis of gene expression databases [–48] shows statistically significant downregulation of OXTR in infection-afflicted humans, as compared to healthy individuals. ***P < 0.001. b. Scatter plots showing data from different databases designated by different colors. Dots are the OXTR levels in individual samples. Data obtained from every database showed diminished OXTR upon HIV, SIV, influenza virus, etc., infections, ranging from 47 to 82% downregulation, as compared with healthy control expression level. (OXTR: mean:0.475, SEM: 0.026; mean: 0.798, SEM: 0.019; mean: 0.826, SEM: 0.016; mean: 0.714, SEM: 0.043; mean:0.716, SEM: 0.018)

Fig. 4

Viral transductions or infections do not universally downregulate cell surface receptors. a. qRT-PCR in primary mouse myoblasts transduced in vitro with non-target GFP shRNA vector at MOI = 0.5; TGFBR1 and R2 mRNA levels were not significantly changed upon transduction. b. qRT-PCR in primary human myoblasts transduced in vitro with non-target GFP shRNA and Smad3-targeting vectors at MOI = 0.5; unchanged TGFBR1 expression and elevated TGFBR2 expression were detected in three independent experiments with each cohort. c. Analysis of GEO human population gene expression databases [–48] reveals no significant change of TGFBR1 and R2 in people upon viral infections. Scatter plots of different databases showed little variation in the expression in TGFBR1, but increased variation in TGFBR2 after viral infections and increase in Smad3 expression in some studies (while others showed no significant change in Smad3). (Smad3: mean:1.101, SEM:0.004; mean:1.702, SEM:0.011; Mean: 0.979, SEM: 0.012; Mean: 1.611, SEM: 0.115; mean: 1,042, SEM: 0.023); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. d. Western blotting analysis of OXTR, pERK1/2, and TGF-β receptor 2 in un-transduced primary mouse myoblasts and mouse myoblasts transduced with non-target shRNA lentiviral vectors. Compared with un-transduced cells, virally transduced myoblasts showed reduction OXTR and pERK1/2 protein levels that became more significant with increased time, while TGF-β receptor 2 levels did not change. Three individual western blots were quantified by pixel intensity. OXTR was normalized to actin, and pERK was normalized to ERK. (*P ≤ 0.05; ** P ≤ 0.01, ***P ≤ 0.001)

Fig. 5

Lentiviral transduction decreases myogenic activity in primary mouse myoblasts. a. Representative 20× IF images for Hoechst (blue), Ki67 (red), and Pax7 (green). Percent of proliferative myogenic cells Ki67+/Pax7+ were quantified out of total Hoechst + cell numbers and were found to be significantly decreased after transduced with either empty vector particles or non-target GFP shRNA vectors. b. Number of single positive (Pax7+ or Ki67+) cells out of total Hoechst + cells were quantified and each population was diminished by either empty vector particles or non-target GFP shRNA vectors. (N = 3, ** P ≤ 0.01 ***P ≤ 0.001)