Different exercise modalities have distinct effects on the integrin-linked kinase (ILK) and Ca2+ signaling pathways in the male rat bone

Mechanical loading is essential to maintain optimal skeletal health. Despite the fact that early-life exercise has positive, long-lasting effects on the musculo-skeletal system, the response of the musculo-skeletal system to spontaneous low-impact exercise has been poorly studied. Previously, we identified subtle morphological changes in the femoral diaphysis of exercised animals compared to nonexercised controls. We hypothesized that significant changes in gene expression of cells should precede significant measurable phenotypic changes in the tissues of which they are part. Here, we employed RNA-Seq to analyse the transcriptome of the cortical bone from the femoral mid-diaphysis of prepubertal male Sprague-Dawley rats that were assigned to control (CON); bipedal stance (BPS); or wheel exercise (WEX) groups for 15 days. We identified 808 and 324 differentially expressed transcripts in the BPS and WEX animals respectively. While a number of transcripts change their levels in an exercise-specific manner, we identified 191 transcripts that were differentially expressed in both BPS and WEX. Importantly, we observed that the exercise mode had diametrically opposite effects on transcripts for multiple genes within the integrin-linked kinase (ILK) and Ca2+ signaling pathways such that they were up-regulated in BPS and down-regulated in WEX. The findings are important for our understanding of possible ways in which different exercise regimens might affect bone when normal activities apply mechanical stimuli during postnatal growth and development.


Introduction
Associational studies have established positive and longlasting effects of early-life exercise in people who engaged in competitive sport as youths or adolescents (Kannus et al. 1995;Karlsson et al. 1995). Due to the intense level (s) of training required to achieve this competitive rank, such activity is unsuitable as a health recommendation for a majority of any community. However, despite the fact that mild or moderate early-life exercise does not negatively affect bone (Firth et al. 2004) or joint development (Van Weeren and Barneveld 1999;Jones et al. 2003), the long-lasting effects of this form of exercise on the musculoskeletal and other body systems have been poorly described. Indeed, morphological studies into the effects of early exercise are further complicated by the fact that the effects on bone phenotype in young animals might be obscured by the normal musculoskeletal growth, and thus be difficult to detect.
Integrating physiological, morphological, and molecular responses of bone to spontaneous exercise is vital for exercise to be advocated as an effective strategy for maintaining/improving bone health parameters. Previously, we identified subtle morphological changes in the femoral diaphysis of prepubertal male rats that were allowed to undertake wheel running (WEX) or had to rise to an erect bipedal stance (BPS) when feeding, for a period of only 15 days (Sontam et al. 2015). These exercise programs were chosen to minimize the confounding effects of stress, and the exercise period was limited to 15 days to ensure that the manipulation was specific to the prepubertal period.
We hypothesized that significant changes in gene expression of cells should precede significant measurable phenotypic changes in the tissues of which they are part. Here, we characterize and compare the global gene expression profile changes in the diaphysis of BPS or WEX rats to those of conventionally housed rats (Control; CON). We identify shared and exercise-specific changes to pathways involved in mechanotransduction, energy homeostasis and cell-cell junction signaling. We show diametrically opposite effects on transcripts for multiple genes within the integrin-linked kinase (ILK) and Ca 2+ signaling pathways such that they were up-regulated in BPS and down-regulated in WEX.

Study design
Tissues that were analysed in this paper were collected simultaneously with those reported in Sontam et al. (Sontam et al. 2015). All animal work was performed under approval R1068 (Animal Ethics Committee, University of Auckland). Twenty-four weanling (21 day old) male pups were obtained from four time-mated Sprague-Dawley rats and assigned to three exercise groups (n = 8): control (CON), bipedal stance (BPS), and wheel exercise (WEX) as previously described (Sontam et al. 2015). Briefly, body composition of the animals was determined before the start of exercise, on day 20 (D 20 ) using dual energy X-ray absorptiometry (DXA, Lunar Hologic, GE, Waltham, MA). The animals were assigned to one of the three exercise groups such that the body weight and total fat percentage were not significantly different between the groups at the outset of the experimental protocol. All animals had ad libitum access to food (Diet 2018, Teklad Global 18% Protein Rodent Diet, Harlan, Teklad) and water.
All groups were pair-housed. CON group rats had unrestricted movement within their cage. BPS animals had to fully extend their tibiotarsal and femorotibial joints to reach their food which was 220 mm above the cage floor. WEX rats could voluntarily access a zero-resistance activity wheel. Food and water intake was measured daily from D 20-27 and on alternative days from D 27-35 . The trial period lasted from D 21 to D 35 . A further DXA scan was undertaken at the end of the exercise period (D 35 ).

Tissue collection
On D 36 , animals were culled by decapitation under isoflurane anesthesia following an overnight fast. Epididymal and retroperitoneal fat pads were collected and weighed. The left femur was immediately dissected from the surrounding soft tissue and was sectioned into proximal, middle, and distal thirds, which were snap-frozen in liquid nitrogen and stored at À80°C; the right femur was harvested, cleaned, and stored in 70% ethanol for microcomputed tomography (lCT) analysis.

Read alignment and differential gene expression analysis
Four biological replicates from CON and five biological replicates each from BPS and WEX groups were included in the differential gene expression analysis. Sequenced read quality was assessed (FastQC; (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)). Phred quality scores were >28 and trimming was not necessary. Sequenced reads were aligned to the rat transcriptome (reference sequence NCBI version Rnor_5.0) using TopHat (version 2.0.12) by providing the GTF file and parametersr 200 -G (Trapnell et al. 2009). The concordant pair alignment rate was 92.6-93.9% with 7.0-8.9% multiple alignments and 1.2-1.7% discordant alignment rates.

Pathway analysis
Differentially expressed transcripts were loaded into Ingenuity Pathway Analysis (IPA Winter Release 2014, IPA, QIAGEN Redwood City, www.qiagen.com/ingenuity). The dataset was filtered using the following attributes: Pvalue = 0.05; the user dataset was used as the reference set; and Rattus norvegicus was specified as the species. IPA identified pathways with significant enrichments and ranked them according to P-values derived from a right tail Fisher exact test.

Results
The phenotypic characteristics of animals within the BPS, CON, and WEX groups have been previously described by our group (Sontam et al. 2015). Briefly, we showed that the diaphysis of femurs within the WEX group had a significantly higher cortical cross-sectional thickness and percent closed porosity than the CON group (Sontam et al. 2015). We showed that the effects of exercise on these changes were independent of caloric intake, body weight, or measures related to stress. The initial changes that we saw led us to hypothesize that RNA expression patterns may be altering in response to the different exercise modalities. Therefore, we undertook a retrospective analysis of the RNA profiles in the femurs of these animals.

Differential gene expression
There were 324 differentially expressed transcripts in the mid-diaphysis of the WEX animals when compared to CON (False Discovery Rate [FDR] adjusted Pvalue < 0.05). The gene transcripts that were up-regulated in the WEX rats were enriched for Gene Ontology (GO) terms associated with skeletal system development and homeostasis (P-value ≤ 0.0218; Table 2A). These GO terms included genes that are involved in bone metabolism (e.g. Bmp2, Cat, Dkk3, Ephb3, Epha4, Fap, Fzd1, Gdf10, Gnas, Lrrc17, Myoc, Nfib, Ostn, Tbx18, Twist1, and Wnt5a). The gene transcripts that were down-regulated in the mid-diaphysis of the WEX rats were enriched for GO terms associated with muscle and blood (P-value ≤ 6.44E-07; Table 2B). There were 808 differentially expressed transcripts (DETs) in the mid-diaphysis of the BPS animals when compared to CON (False Discovery Rate [FDR] adjusted P-value < 0.05). The gene transcripts that were up-regulated in the BPS group were associated with GO terms related to muscle cells and energy metabolism (P-value ≤ 5.10E-18; Table 3A). The gene transcripts that were down-regulated in the BPS animals were enriched for GO terms associated with the immune system (P-value ≤ 5.20E-12; Table 3B).
There were 191 gene transcripts that were differentially regulated in both forms of exercise. Within this common set of gene transcripts, 51 were up-regulated in both conditions and were enriched for GO terms associated with skeletal system development. Fifty-one transcripts were down-regulated in both forms of exercise and were enriched for immune system processes. The remaining 89 gene transcripts were enriched for GO terms related to muscle contraction. The expression changes of these 89   gene transcripts were directionally dependent upon the exercise modality: specifically, these 89 transcripts were up-regulated in the BPS and down-regulated in the WEX group (Fig. 1). In order to determine if any of the common 191 differentially regulated transcripts were influenced by the magnitude of the exercise performed, the WEX animals were divided into two subgroups based on their indi-vidual total running distance within a monitored 36 hour period (D 33 ). The mean (AESEM) distances run by the low (2130 AE 575.55 m) and high (3888.04 AE 825.9 m) running subgroups were significantly different (P-value: 0.04). Differential gene expression analysis between the high and low running subgroups identified six transcripts (LOC100360843, Ankrd2, Myh7, Myl3, Myoz2, and Tnnt1) that were significantly changed in the high, and two transcripts (Fkbp5 and Ccl21) that were significantly changed in the low running subgroups.

Pathway analysis
Ingenuity pathway analysis revealed that the WEX-affected transcripts were significantly enriched in pathways for epithelial adherens junction signaling, calcium signaling, integrin linked kinase (ILK) signaling, cellular effects of sildenafil, and remodeling of epithelial adherens junction signaling (Table 4). By contrast, BPS-affected transcripts were significantly enriched in calcium signaling, ILK signaling, cellular effects of sildenafil, oxidative phosphorylation, and mitochondrial dysfunction pathways (Table 4).
Ca 2+ , ILK and sildenafil pathways responded to both BPS and WEX. However, the genes that responded to BPS and WEX within these pathways exhibited exercisedependent changes in transcript levels. For example, the transcript levels of nine genes in the ILK signaling pathway responded to both treatments (Fig. 2). Of these, eight transcripts were up-regulated in BPS and down-regulated in WEX. Only one gene transcript (Actg2) was up-regulated in both treatments (Fig. 2).
Transcripts associated with Ca 2+ signaling pathways also exhibited exercise-dependent changes (Fig. 3) for 10 genes. Again, these genes showed diametrically opposite responses to the exercise modalities being up-regulated in BPS, and down-regulated in WEX (Fig. 3).   -- Trdn↑ Trdn↓  in the mid-diaphysis of prepubertal male rats whose bone micro-architecture and morphology showed minor changes in response to exercise. We identified and characterized several transcripts whose expression levels were affected by the exercise undertaken. The observation that the two exercise modalities elicited a diametrically opposite response for a subset of transcripts is particularly relevant given the perception that physical exercise promotes long and short-term bone health. Indeed, our results are consistent with earlier observations that the form and intensity of the exercise is critical for the bone-specific response that is elicited (e.g. [Milgrom et al. 2000]). Direct comparisons regarding the activity type and activity cycles and therefore the strain magnitude and frequency required to elicit these changes are not currently possible, as there are no biomechanical models for these exercises in the rat. Moreover, determination of the actual exercise-dependent changes in the strain environment of bone cannot currently be empirically determined (Meakin et al. 2014). We contend that future studies should characterize the exercise-dependent changes that occur at specific regions and to specific cells within bones, and at different stages of growth and development, in order that the full benefits of exercise can be harnessed to improve or maintain bone health.