ABSTRACT

Objective

Spinal muscular atrophy (SMA) is the most common neurodegenerative disease caused by the absence or insufficiency of the survival motor neuron (SMN) protein. Human SMN1 (hSMN1) produces fully functional SMN protein but hSMN2 produces only about 10% functional protein. Deletion or mutation in hSMN1 gene leads to SMA, while the hSMN2 copy number modifies disease severity. Increasing hSMN2 expression has emerged as a potential therapeutic approach. In this study, we investigated the effect of growth hormone (GH) on hSMN2 promoter activity using a reporter in Chinese hamster ovary (CHO) cells.

Methods

Three different hSMN2 promoter regions (588 bp, 1036 bp and 1705 bp) were used to show the effect on gene expression of reporter response to GH in this study. Promoters were amplified by polymerase chain reaction (PCR) and cloned into the pGL3 luciferase reporter vector. The ligation reactions were transformed into DH5α cells and positive colonies containing specific hSMN2 promoter inserts were confirmed by PCR with hSMN2-primers. The plasmids carrying hSMN2 promoters were transfected into CHO cells. After transfection, the cells were treated with GH for 24 hours and luciferase activity was measured to assess promoter activity.

Results

All hSMN2 promoter constructs responded to GH. The 1036 bp promoter construct showed the highest luciferase expression upon GH treatment. However, the 1705 bp promoter construct exhibited reduced gene expression compared to the control vector treated with GH.

Conclusion

These findings suggest that GH can modulate hSMN2 expression in hSMN2 promoter dependent manner. GH may be a candidate hormone for SMA treatment by enhancing hSMN2 expression.

Introduction

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease characterized by degeneration of spinal cord motor neurons associated with proximal muscle weakness and muscular atrophy (1). SMA affects 1 in 6,000 to 1 in 10,000 individuals worldwide (2). Based on the age of onset and severity of the clinical course, childhood-onset SMA can be classified into four types (types I-IV). The most severe and most common (45%) type is type 1, which presents in infancy, and the mildest form is type 4, which is present in adults (3). Muscle weakness and impaired mobility are characteristic features of SMA (4). SMA is caused by a deficiency of the survival motor neuron (SMN) protein (1). The gene responsible for encoding the SMN protein maps to the reverse duplication site on 5q11.2-q13.3 and is called the SMN gene (5). In humans, there are two copies of the SMN gene, identified as human SMN1 (hSMN1) (telomeric) and hSMN2 (centromeric) and this is unique to Homo sapiens (6). All forms of SMA result from homozygous loss of the hSMN1 gene due to gene deletion, conversion or mutation (7). Consistent with this gene duplication being a very recent evolutionary event, the hSMN1 and hSMN2 genes share more than 99.8% sequence homology over a 30 kb segment containing the entire coding region (8). hSMN1 is composed of nine exons, 1, 2a, 2b, 3, 4, 5, 6, 7 and 8 (untranslated exon 8) encoding a 294 amino acid (aa) protein with a molecular weight of 38 kDa (9). hSMN1 gene is transcribed into a full-length (FL) messenger RNA (mRNA). However, hSMN2 is primarily transcribed into alternatively and naturally spliced mRNA lacking exon 7. This alternative splicing is caused by a silent mutation (C to T) in exon 7 of the hSMN2 gene, which results in the loss of an exon splicing enhancer or the creation of an exon splicing repressor (1). The hSMN2 gene mainly produces 90% transcripts lacking exon 7 and 5-10% wild type transcripts (10). When the hSMN1 gene is deleted or mutated, the hSMN2 gene cannot produce sufficient levels of functional SMN protein, resulting in SMA. All SMA patients have one or more copies of hSMN2 gene and there is an inverse correlation between SMA severity and hSMN2 copy number. The relationship between disease severity and copy number for hSMN2 correlates with an increase in the FL SMN produced by each additional hSMN2 gene (11). Increasing expression level of the hSMN2 gene is considered an important strategy in the treatment of SMA.

Growth hormone (GH) has long been known to be a regulator of growth and sugar-fat metabolisms, but mechanisms of the transcription regulation by GH for some specific genes, such as hSMN2, are not described. GH binds to two GH receptors (GHR) and this ternary complex activates GHR-associated Janus kinase 2 (JAK2), which in turn phosphorylates tyrosines residues in itself, on the GHR and on intracellular proteins. Phosphorylated tyrosines on the receptor form docking sites for a number of signaling proteins, including members of the signal transducers and activators of transcription (STAT) family. Phosphorylated STAT proteins are released from the receptor and then they are dimerized, migrate to the nucleus and play an important role in the regulation of gene transcription (12). The role of the JAK/STAT signaling pathway in the regulation of hSMN2 expression has also been demonstrated (5). STAT5 transcription factor in the STAT family plays an important role in the JAK2/STAT5 pathway. The PRL JAK2/STAT5 pathway is known to be involved in the regulation of hSMN2 gene expression (5). GH is plays a major role in activation of STAT5 but there is no information about increased hSMN2 gene expression by GH through the JAK-STAT5 signaling pathway in humans.

There is currently no cure for SMA. There are only treatments that slow the progression of disease severity and reduce symptoms. Recent studies have indicated that up-regulating hSMN2 gene expression may be a possible treatment for SMA.

Previous in vivo studies have suggested that GH may influence SMN expression through STAT5 pathway activation. In particular, MacKenzie et al. (13) demonstrated that systemic administration of human GH (hGH) in severe SMA mouse models increased SMN protein levels in the brain and spinal cord, improved disease phenotype, and significantly prolonged survival. These results identified hGH as a potential therapeutic compound acting via STAT5 signaling (13). Building upon these findings, our study focused on the direct transcriptional regulation of the hSMN2 promoter by GH in a cell culture system, aiming to provide mechanistic evidence for the promoter-level responsiveness of SMN2 to GH.

In this study, our purpose was to determine whether or not GH specifically increased the expression level of luciferase gene for the reporter vectors containing three different promoter regions of hSMN2 gene in Chinese hamster ovary (CHO) cells.

Methods

Bioinformatic Analysis

The promoter regions of hSMN1 and hSMN2 genes were analyzed using data from the National Center of Biotechnology Information (NCBI) and The Eukaryotic Promoter Database (EPD). The promoter sequence of the two genes was compared and differences were determined using the bioinformatic tool, VectorBuilder (VectorBuilder Inc. 1010 W 35th Street, Suite 515 Chicago, IL 60609, USA Tel: +1 800-517-2189; https://en.vectorbuilder.com/). STAT5 transcription sites in promoter region of hSMN2 gene were analyzed using EPD. A restriction enzyme map in the hSMN2 promoter region was analyzed using NEBcutter 3.0 (a tool provided by New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938‑2723, USA; Tel: +1 978‑927‑5054; Email: [email protected]; Website: https://www.neb.com/). Restriction enzyme cut sites were determined for cloning, based on restriction enzyme analysis of hSMN2 promoter and the pGL3 vector cloning site. Restriction enzymes used in the study were NheI and XhoI (New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938‑2723, USA). Primers for hSMN2 promoter regions were designed specifically and NheI and XhoI restriction sequences were added to 5’ site of the primers. The primers for hSMN2 promoter regions were analyzed for Tm and GC values using OligoAnalyzer™ Tool (Integrated DNA Technologies, Inc., Coralville, IA 52241, USA). Specificity and size of amplicons were also checked with NCBI Primer Blast Tool.

Molecular Biology Studies

DNA Isolation and PCR

Genomic DNA was isolated from blood using a salting out technique (14). DNA concentration and purity were measured with a NanoDrop (Thermo Fisher, USA) and DNA with an A260/A280 ratio between 1.8-2.0 was used for polymerase chain reaction (PCR). The PCR reaction for promoter regions 1 and 2 of hSMN2 was prepared in a total volume of 25 µL and composed of 1x10X PCR Buffer, 1.5 mM MgCl2, 200 μM dNTP mixture, 0.5 µM hSMN2 Forward1 or 2 primer, 0.5 µM hSMN2 reverse primer, 500 ng genomic DNA, 1U Taq polymerase (Promega, USA). PCR amplification was performed using Thermal Cycler (Bio-Rad T100 96-Well, US) under the following conditions: initial denaturation: 94 °C 2 min (1X); first cycle: 94 °C 30s, 61 °C 30s, 72 °C 1 min (5X); following second cycle: 94 °C 30s, 65 °C 30s, 72°C 1 min. (25x); final extension: 72 °C 10 min. The long-range PCR for hSMN2 promoter region 3 was performed in a total volume of 50 µL and composed of 1x PCR Buffer with MgCl2, 200 μM dNTP mixture, 0.5 µM hSMN2 Forward3 primer, 0.5 µM hSMN2 Reverse primer, 500 ng Genomic DNA, 1U Taq polymerase (Takara, Japan). PCR amplification was performed using Thermal Cycler (Bio-Rad T100 96-Well, US) under the following conditions: initial denaturation: 94°C 2 min (1X), 94 °C 30s, 65 °C 30s, 72 °C 2 min (5x); following cycle: 94 °C 30s, 68 °C 30s, 72 °C 2 min, Final extension: 72 °C 10 min. PCR products were analyzed by 1% agarose gel electrophoresis and the ethylene bromide-stained gel was visualized using the Gel Imaging System (Biolab, UK).

PCR Purification

PCR purification was performed using the High Pure PCR Product Purification Kit (Roche, Switzerland). Concentration of the purified PCR products was measured by NanoDrop (Thermo Fisher, USA).

Double Digestion

Double cut was performed for the hSMN2 promoter PCR products and pGL3 vector using 25 µL PCR product (2 µg) or pGL3 vector (2 µg), 5 µL 10X Buffer (rCutSmart), 1 µL XhoI (20U), 1 µL NheI (20U) with a total volume of 50 µL. The digestion reactions were incubated at 37 °C for 4 hours (Bacterial Incubator, Binder, Germany). Digested pGL3 vectors and PCR products were purified as previously described. Concentrations of double cut PCR products and pGL3 vector were measured and analyzed by agarose gel electrophoresis.

Ligation

The double cut hSMN2 PCR promoter products were ligated into double cut pGL3 vector using T4 DNA ligase (3U, Promega, USA). The ligation reactions were performed in 1/1, 1/3 and 1/5 ratios and incubated at +4 °C overnight. Restriction enzymes were inactivated 80 °C and 65 °C for 20 min respectively. Then, transformation was performed using heat shock method with 5 μL ligation product and 100 μL DH5α competent cells (Takara, Japan). Transformed products were plated on bacterial plates with ampicillin and incubated at 37 °C overnight (Bacterial Incubator, Binder, Germany).

Colony PCR

Direct colony PCR was performed to determine insertion of promoter regions of hSMN2 gene in the pGL3 vectors on colonies. The transformed colonies on the plates were transferred into tubes containing 10 μL distilled, DNA-free water (dwater), from which 4 μL bacterial aliquot were taken into sterile tubes. The remaining 6 μL were incubated at 95 °C for 10 minutes. For direct colony PCR content: 2 μL template, 1.5 μL 10X PCR Buffer, 1.5 mM MgCl2, 200 μM dNTP, 0.4 μM forward primer for vector, 0.4 μM reverse primer for vector, 0.5U Taq DNA Polymerase (Promega), 7.5 μL dwater. PCR amplification was performed using Thermal Cycler (Bio-Rad T100 96-Well, US) under the following conditions: initial denaturation: 94 °C 2 min (1X), 94 °C 30s, 55 °C 30s, 72 °C 90 sec (25X), final extension: 72 °C 5 min. PCR products were analyzed by 1% agarose gel electrophoresis and visualized using the Gel Imaging System (Biolab, UK).

Plasmid Isolation

Positive colonies were cultured overnight at 37 °C and the plasmid isolation was performed using a Genopure Plasmid Isolation Kit (Roche, Switzerland) following manufacturer protocol and DNA concentrations were measured.

Specific PCR for plasmids obtained from positive colonies. PCR was performed using 100 ng/μL plasmids, 1.5 μL 10X PCR Buffer, 1.5 mM MgCl2, 200 μM dNTP, 0.5 μM forward primer (hSMN2 forward 1 or hSMN2 forward 2 or hSMN2 forward 3), 0.5 μM reverse primer (hSMN2 reverse), 1U Taq DNA Polymerase and 8.5 μL dwater. PCR amplification was performed using Thermal Cycler (Bio-Rad T100 96-Well, US) under the following conditions: Initial denaturation: 94 °C 2 min, 94 °C 30s, 67 °C 30s, 72 °C 90 sec (30x), Final extension: 72 °C 5 min.

Cell Culture Studies

CHO cells were cultured in T25 flask containing High Glucose with L-Glutamine (500 mL, PAN-Biotech, Germany), 10% Fetal Bovine Serum, heat inactivated (500 mL, Wisent Inc, Canada) and 500 μL penicillin + streptomycin and incubated at 37 °C with 5% CO₂ (Mammalian cell culture incubator, Binder CB150. Germany).

Transfection

CHO cells were plated into 24 well plates containing 0.05x10⁶ cells. After 24 hours incubation, transfection was done using TransIT®-2020 Transfection Reagent (Mirus, USA) based on company protocol. Transfection was performed by 400 ng GHR, 400 ng STAT5 and 300 ng different reporter constructs containing different promoter regions of the hSMN2 gene. Transfected CHO cells were incubated at 37 °C with 5% CO₂ for 24 hours.

GH Treatment

After 24 hours of transfection, the medium of the transfected CHO cells was removed and the cells were washed 3 times with DMEM. The transfected cells were starved with 0.5 mL DMEM for 1 hour at 37 °C in a 5% CO₂ incubator. The transfected cells were treated with GH (Genotropin Goquick, 5.3 mg/mL); (1000 ng/mL for each well) for 24 hours at 37 °C with 5% CO₂.

Cell Culture Lysis and Luciferase Assay

Cell Culture Lysis Buffer 5X Reagent (Promega, USA) was used for the lysis of cells. Medium in 24-well plate was removed and the cells were washed 3 times with cold 1X PBS. 100 μL Luciferase Cell Culture Lysis Reagent (1X) was added to each well and shaken for 15 min. The cell lysates were transferred into sterile centrifuge tubes and luciferase activity was performed using Promega Luciferase Assay System protocol. 100 μL of luciferase substrate and 20 μL of cell lysate were added into each well of a 96-well plate and luciferase activities were measured on EnSpire Multimode Plate Reader (PerkinElmer Inc., USA).

Statistical Analysis

Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) for Windows, version 30.0.0.0 (IBM Inc., Armonk, NY, USA). Normality of the data was assessed using the Shapiro-Wilk tests. A one-way ANOVA was performed to compare the luciferase activity among the four experimental groups. A post-hoc test (Tukey) was conducted to identify specific group differences. A p-value<0.05 was considered statistically significant.

Results

STAT5 binding sites in the promoter region of hSMN2 gene were analyzed using the EPD tool and binding sites were located at -26, -334, -523, -750, -1631, -1686 from transcription start site. NheI and XhoI restriction sites were chosen for cloning based on pGL3 vector cloning sites and no presence in the hSMN2 promoter. Three hSMN2 promoter regions from transcription start site were amplified successfully by specific hSMN2 promoter primers shown in Table 1. Amplified PCR products were analyzed by agarose gel electrophoresis and size of PCR products was 588 bp, 1036 bp and 1705 bp fragments, respectively (Figure 1).

The amplified hSMN2 PCR promoter products and pGL3 vector were digested by NheI and XhoI restriction enzymes. The digested PCR products were ligated into NheI and XhoI sites located in front of luciferase gene in pGL3 vector producing three pGL3-hSMN2 promoter1, pGL3-hSMN2 promoter2 and pGL3-hSMN2 promoter3 constructs. In order to determine hSMN2 promoter insert in the transformed colonies on amp plates, colony PCR was performed successfully using pGL3 vector primers shown in Table 1 and Figure 2 shows the agarose gel electrophoresis results of colony PCR. The size of PCR products including vector sequence were 788 bp, 1236 bp and 1905 bp respectively and these were the expected sizes for hSMN2 promoters plus part of vector.

In order to confirm specific hSMN2 promoter inserts in positive colonies, plasmids were isolated from cultured bacteria and they were amplified by specific hSMN2 primers and PCR-agarose gel electrophoresis analysis were performed (data not shown). Results showed that inserts in plasmids were specific hSMN2 promoter sequences and the sizes were correct.

In order to determine the effect of hGH on hSMN2 promoters driving luciferase gene expression, transfected CHO cells expressing pGL3 alone, pGL3-hSMN2 promoter1, pGL3-hSMN2 promoter2 and pGL3-hSMN2 promoter3 were treated with hGH and then their luciferase activities were measured. Luciferase results showed that GH strongly induced luciferase reporter gene expression for all reporter constructs driven by hSMN2 promoters compared to luciferase expression of pGL3 vector alone, with or without GH treatment. Although the pGL3-hSMN2 promoter2 construct induced by hGH produced the highest level of luciferase gene expression, the pGL3-hSMN2 promoter3 construct suppressed luciferase gene transcription, as shown in Figure 3.

Statistical analysis was performed by assessing the normality of the data using the Shapiro-Wilk test. To assess the normality of each group’s data, the Shapiro-Wilk test was conducted. All groups showed p-values greater than 0.05 [pGL3 vector (+): p=0.824, pGL3-hSMN2 Promoter 1 (+): p=0.567, pGL3-hSMN2 Promoter 2 (+): p=0.943, pGL3-hSMN2 Promoter 3 (+): p=0.846], indicating that the data were normally distributed. However, due to the small sample size (n=3 per group), the results of the normality tests should be interpreted with caution. A one-way ANOVA was performed to compare the luciferase activity among the four experimental groups [(1) pGL3 Vector (+), (2) pGL3-hSMN2 Promoter 1 (+), (3) pGL3-hSMN2 Promoter 2 (+), (4) pGL3-hSMN2 Promoter 3 (+)]. The analysis revealed a statistically significant difference between the groups (p=0.002). Post-hoc analyses using Tukey’s HSD test revealed significant differences between Group 1 and Group 2 (p=0.007), Group 1 and Group 3 (p=0.001), and Group 3 and Group 4 (p=0.030). No significant differences were found between the other group pairs. These results are presented in Table 2.

Discussion

SMA is an inherited autosomal recessive neurodegenerative disease presenting with variable phenotype and is characterized by the loss of motor neurons from the anterior horn cells of the spinal cord, resulting in progressive muscle loss and respiratory failure (7). Most cases of SMA (95%) have a homozygous deletion in the hSMN1 gene on chromosome 5q13.

SMA is one of the most common autosomal recessive neuromuscular disorders. However, clinical heterogeneity in disease phenotype depends on hSMN1 gene (Telomeric) and hSMN2 (Centromeric) genes, specifically the varying copy number of hSMN2. There is only one base difference between hSMN1 and hSMN2 genes and C residue at exon 7 in hSMN1 is converted to T residue in SMN2 gene (c.840C>T) disrupting the exogenic splicing enhancer (1). C->T substitution causes abnormal splicing resulting in removal of exon 7 (1). Exon 7 deleted mRNA gives truncated non-functional protein. SMA patients lacking hSMN1 are dependent on the amount of residual hSMN2 functional SMN protein for alpha motor neuron function. Several reports have shown that there is a strong positive correlation between SMA phenotype severity and the number of copies of hSMN2 gene. Patients carrying high copy number of hSMN2 show milder SMA (11).

There are several approaches to treatment of SMA, one of which increases hSMN2 gene expression. There are numbers of studies that have been shown to increase hSMN2 expression levels (1, 5, 7, 13, 15, 16). Andreassi et al. (7) investigated the effect of 4-phenylbutyrate (PBA) treatment on hSMN2 gene expression in fibroblast cell cultures obtained from SMA patients and PBA increased FL hSMN2 transcript levels and SMN protein in cells from patients with all SMA types (Type I, II and III). Grzeschik et al. (1) showed the effect of hydroxyurea (HU) treatment on hSMN2 gene expression in lymphoblastoid cell lines derived from SMA patients and HU was shown to increase the FL hSMN2 transcript ratio in a dose and time-dependent manner. In addition to this, a significant increase in SMN protein levels and significantly increased nuclear gem (Gemini of Cajal bodies), which are SMN-containing nuclear structures were shown with treatment using HU (1). Biondi et al. (15) reported that NMDA receptor activation accelerated motor neuron maturation, reduced apoptosis and increased hSMN2 gene expression in SMA model mice. It was reported that GH induced SMN expression in an SMA animal model (16). Previous studies have shown that GH activates the JAK/STAT pathway (15). However, the direct effect of GH on hSMN2 promoter activity has not been previously reported in mammalian cells. Our study is first study to show that transfected cells incubated in the presence of hGH strongly increased hSMN2 promoter driving gene expression of luciferase gene of construct in mammalian cells.

Although it has been reported that GH can regulate motor neuron function through the JAK/STAT pathway (5), the present study fills an important evidence gap by demonstrating the potential for GH to be used as a therapeutic target in the treatment of SMA.

Our results, showing a strong and specific activation of the hSMN2 promoter by GH in vitro, are consistent with the findings of MacKenzie et al. (13) who reported that GH treatment increased SMN protein levels and extended survival in severe SMA mouse models. While their work demonstrated the therapeutic relevance of GH in vivo, our data provides mechanistic support at the transcriptional level by confirming that GH-linked signaling pathways can directly activate the hSMN2 promoter. Together, these complementary studies strengthen the rationale for further investigation of GH or GH-related STAT5 activators as candidate therapeutic agents in SMA. However, translation from promoter-reporter assays to clinical application requires additional validation in motor neuron-derived cells, in vivo studies and eventually patient-based models.

Our hSMN2 promoter studies showed that hSMN2 promoter regions exhibited different levels of transcriptional activity in response to hGH treatment. pGL3-hSMN2 Promoter 2 driving construct exhibited the highest luciferase activity among the hSMN2 promoters, as shown in Figure 3. However, pGL3-hSMN2 Promoter 3 driving reporter constructed lowered promoter activity, indicating a potentially suppressor regulatory role in promoter region. Luciferase activity of the hGH-treated pGL3-hSMN2 promoter 1 region increased 191.6-fold, GH-treated pGL3-hSMN2 promoter 2 region increased 348-fold and GH-treated pGL3-hSMN2 promoter 3 region increased 133-fold compared to GH-treated plasmid alone, as shown in Table 3. The different transcriptional activities of the three hSMN2 promoters may indicate the existence of enhancers and suppressor sequences located at the promoter regions for binding sites for transcription factors activated by GH. The 669 bp hSMN2 promoter region between residues 1036 and 1705 contains a suppressor sequence. Addition to in vitro studies on CHO cells, GH-induced hSMN2 expression studies should be performed in vitro in human motor neurons cell or human fibroblast cell cultures obtained from patients with different type of SMA or in in vivo models.

Study Limitations

One of the limitations of our study was that we did not perform an electrophoretic mobility shift assay to demonstrate the binding of GH-induced STAT5 or other transcription factors to the SMN2 promoter. Due to limited funding, we were unable to utilize radioactive labeling of the SMN2 promoter, and we also lacked the necessary equipment to carry out this analysis. Therefore, we could not directly assess the transcription factors involved in the regulation of SMN2 expression.

Conclusion

These findings suggest that GH may be a potential therapeutic target in the treatment of SMA, but this needs to be confirmed in vivo in large animals. It is hoped that this study will stimulate investigation of a new therapeutic approach for SMA by demonstrating the effects of GH on hSMN2 expressions. Our findings are among the first to identify the effects of GH on hSMN2 promoter regions and this may provide a basis for further studies.