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Age-related resistance of skeletal muscle-derived progenitor cells to SPARC may explain a shift from myogenesis to adipogenesis

Age-related resistance of skeletal muscle-derived progenitor cells to SPARC may explain a shift from myogenesis to adipogenesis


Abstract

Aging causes phenotypic changes in skeletal muscle progenitor cells (SMPCs) that lead to the loss of myogenicity and adipogenesis. Secreted protein acidic and rich in cysteine (SPARC), which is secreted from SMPCs, stimulates myogenesis and inhibits adipogenesis. The present study aimed to examine whether changes in SPARC expression, its signaling pathway, or both are involved in age-related phenotypic changes in SMPCs. SPARC expression levels were comparable in SMPCs derived from young and old rats. However, when SPARC expression was reduced by a SPARC-specific siRNA, SMPCs from young rats showed reduced myogenesis and increased adipogenesis. In striking contrast, old rats showed little changes in these functions. Recombinant SPARC was effective in inhibiting adipogenesis and promoting myogenesis of SMPCs from young rats but had no effect on SMPCs from old rats when endogenous SPARC levels were reduced by the SPARC-siRNA. Further, the level of integrin α5, a subunit of the putative SPARC receptor, was decreased in SMPCs from old rats, and its inhibition in SMPCs from young rats by siRNA reduced adipogenesis in response to SPARC. These results suggest that, although SPARC plays a role in regulating SMPC function, SMPCs become refractory to the action of SPARC with age. Our data may explain an age-related shift from myogenesis to adipogenesis, associated with sarcopenia.

Introduction

Aging impairs organ function. Human body mass, of which skeletal muscle is the most abundant tissue, declines with age [1]. The decline in skeletal muscle (sarcopenia) is defined clinically by a loss of lean muscle mass and impairment of its function [2]. Adipose tissue infiltration is often observed in skeletal muscles of the elderly [3], and this intramuscular adipose tissue (IMAT) causes poor physical performance [4] and plays a role in insulin resistance and obesity [5]. Therefore, treating these conditions as well as sarcopenia will depend on a better understanding of the mechanism of IMAT accumulation.

Skeletal muscle contains several types of SMPCs, including satellite cells that possess almost exclusively myogenic potential [6]. Adult mouse SMPCs spontaneously differentiate into adipocytes [7] and their adipogenic potential increases with age [8]. Vettor, et al. postulate that SMPCs form IMAT [9], thus suggesting that alterations in the differentiation of SMPCs cause sarcopenia. Differentiation of SMPCs is greatly affected by their microenvironment [10, 11]. For example, degradation of basal lamina, a major component of the SMPC niche, promotes adipogenic differentiation of SMPCs [11]. Basal lamina contains several extracellular matrix (ECM) proteins such as collagen, laminin, fibronectin and proteoglycans. ECM proteins function as structural supports for SMPCs and bind growth factors that regulate SMPC function [12].

SPARC (also known as osteonectin or BM-40) is a nonstructural, matricellular glycoprotein that is secreted into the niche and functions in cell adhesion, angiogenesis, growth factor binding, and cell differentiation [13, 14]. SPARC inhibits adipogenesis of preadipocytes by enhancing β-catenin signaling [15]. It also promotes myogenic differentiation of C2C12 and MM14 myoblasts [16, 17]. SPARC is expressed by SMPCs [18] and may regulate SMPC function in an autocrine manner. SPARC-null mice exhibit early-onset cataracts [19], osteopenia-like decline of bone formation [20], increased obesity, and decreased skeletal muscle mass [21]. These phenotypes are typically observed in old mice.

In the present study, we examined whether changes in SPARC expression or signaling are involved in age-related phenotypic changes during the differentiation of SMPCs.

Results

SMPCs express SPARC regardless of their differentiation state during myogenesis and adipogenesis

SMPCs express SPARC [18] and can differentiate into myogenic and adipogenic cells [7]. To examine whether the expression of SPARC by SMPCs is differentiation dependent, we immunostained SMPCs derived from young rats by using antibodies specific for MyoD, C/EBPα, myosin heavy chain (MHC), and SPARC (Figure 1). MyoD is a transcription factor that plays a critical role in myogenesis. MyoD-positive cells coexpressed SPARC in SMPC cultures 3 days after plating. MHC-positive myotubes, induced by culturing SMPCs in media containing 2% HS, were also positive for SPARC. Approximately 20% of SMPCs expressed C/EBPα-, a transcription factor involved in adipogenesis, and were stained with Oil Red-O, an indicator of adipogenesis. Both C/EBPα- and Oil Red-O-positive cells expressed SPARC. In all cases, SPARC expression was detected in both non-myogenic and non-adipogenic cells, indicating that SMPC express SPARC regardless of their state of differentiation.

Immunocytochemistry of SPARC in SMPCs

Figure 1. Immunocytochemistry of SPARC in SMPCs. Immunostaining of SPARC during myogenic and adipogenic differentiation of SMPCs from young rats. Cells were co-stained with anti-MyoD and anti-SPARC 3 days after plating or with anti-MHC and anti-SPARC 4 days after exposure to media containing 2% HS. Co-staining with anti-SPARC and anti-C/EBPα or Oil Red O staining was performed 4 days after culturing in the media to induce adipogenic differentiation (ADM). Nuclei were visualized with Hoechst 33258 Dye (blue). White arrowheads indicate double-positive cells (MyoD+/SPARC+, MHC+/SPARC+, C/EBPα+/SPARC+, and Oil Red O+/SPARC+), whereas black arrowheads indicate cells that are only positive for SPARC (MyoD-/SPARC+, MHC-/SPARC+, C/EBPα-/SPARC+, and Oil Red O-/SPARC+). Scale bar = 100 μm.

SPARC expression levels in SMPCs do not change with age

The adipogenic potential of SMPC increases with age [8], which we confirmed here (data not shown). Because SPARC inhibits adipogenesis [15], the age-related increases in the adipogenic potential of SMPCs may be due to a decline in their secretion of SPARC. However, quantitative PCR analysis revealed no differences in SPARC expression levels between SMPCs from young and old rats (Figure 2A). Immunoblotting and immunostaining of SPARC in young and old SMPCs also showed that the levels of SPARC expression in SMPCs does not change with age (Figures 2B and 2C). These results suggest that the increased adipogenic potential of old SMPCs is not caused by a decline in SPARC.



When people speak of contemporary medicine, accuracy plays one of the most crucial roles and human lives are literally dependent on it. Hereby, any researches pertaining to medicine are necessary to comply with the top standards. The issue today is that any conclusions of researches can be shared online and used as a reference without being adequately checked and approved. Mikhail (Misha) Blagosklonny of Oncotarget perfectly understood this challenge and tried to create an alternative solution. That’s how a weekly oncology-focused research journal called “Oncotarget” has been founded back in 2010. The major principle of this journal is related to Altmetric scores that are used as a quality indicator. That assists both readers and authors to verify publications with Altmetric Article Reports that provide “real-time feedback containing data summary related to a particular publication.” Oncotarget website demonstrates a full publications list with corresponding scores above 100 as well as reports discussed above. Mikhail (Misha) Blagosklonny glad to share his new approach and hopes it creates the necessary assistance to anyone, who has interest in oncology.
“A diagnostic autoantibody signature for primary cutaneous melanoma” has the Altmetric score of 594. This article was published back in 2018 by Oncotarget and written by various experts from Hollywood Private Hospital, Edith Cowan University, Dermatology Specialist Group, St. John of God Hospital and The University of Western Australia. The introduction of the study mentions that “recent data shows that Australians are four times more likely to develop a cancer of the skin than any other type of cancer”, and provides an insight on melanoma that “is curable by surgical excision in the majority of cases, if detected at an early stage.”
The publication has got an Altmetric score of 594. Mikhail (Misha) Blagosklonny realizes that most of readers are willing to understand the very meaning of it. Based on the Altmetric website, the score relates to “how many people have been exposed to and engaged with a scholarly output.” Likewise, the article about melanoma, was used for citations in different news articles 69 times. In addition, it was quoted in 2 online blogs, as well as 25 Tweets on Twitter and 1 Facebook post. FOX23 of Tulsa, Oklahoma has headlined their report on July 20, 2018 as “New blood test could detect skin cancer early”, using the main content of Australia study 
Another Oncotarget’s research with a top score of 476, is “Biomarkers for early diagnosis of malignant mesothelioma: Do we need another moon-shot,”. This study has appeared in 60 news stories, 1 online blog post and 6 Twitter posts. The majority of public may have seen a short overview only, however those who visit Mikhail (Misha) Blagosklonny at Oncotarget, do get useful scientific facts. Oncotarget is glad to have the chance to share with online customers this highly appreciated and high-quality information, that is trustworthy and reliable.

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