Motor And Extracellular Matrix Proteins Play A Critical Role In Cleft Palate Formation

By Dr. Chinta SidharthanReviewed by Lily Ramsey, LLMSep 30 2024

Despite the high incidence rate of cleft palate, the underlying factors that contribute to the complex etiology and pathogenesis of cleft palate remain poorly understood. Murine models of cleft palate have been successfully established using retinoic acid, and studies using this model have uncovered the involvement of various proteins in the development of non-syndromic orofacial clefts.

In a recent study published in Scientific Reports, researchers examined the molecular mechanisms of cleft palate using murine models. They reported differential expression of numerous motor and extracellular matrix-related proteins in the cleft palate models.

​​​​​​​Study: Proteomic analysis illustrates the potential involvement of motor proteins in cleft palate development. Image Credit: PeopleImages.com - Yuri A/Shutterstock.com

Background

Cleft palate is a congenital disability that occurs in one in every thousand newborns. While both environmental and genetic factors are thought to influence the risk of cleft palate formation, the exact etiology remains unclear.

Researchers found that elevated levels of retinoic acid, which is an important vitamin A metabolite, resulted in cleft palate formation in mice, which were then used as cleft palate models by various studies.

Studies using these models helped identify the involvement of key proteins such as C-reactive protein, non-syndromic orofacial clefts (NSOFC) protein, apolipoprotein A, and histidine phosphotransfer (HPT) protein, which were potential prenatal diagnosis biomarkers.

Other studies have also identified mutations in various genes, such as the one coding for methylenetetrahydrofolate reductase (MTHFR) and many others associated with cleft palate.

About the Study

In the present study, the researchers used proteomic technology to study the differential expression of proteins among retinoic acid-induced cleft palate murine models and normal controls to decipher the molecular mechanisms of cleft palate development.

The study used female and male-paired mice for breeding. Once pregnancy was confirmed, the experimental group was orally administered retinoid acid, while the control group received no retinoid acid treatment. The palatal tissues of the embryos from each group were collected and analyzed together.

The tissue samples were processed for protein extraction, which included treatment with methanol and methyl tert-butyl ether, as well as mass spectrometric analysis. After digestion with trypsin to break down the proteins, the peptides were labeled with isobaric tags for relative and absolute quantitation or iTRAQ. The peptides were then separated using chromatography.

The mass spectrometry analyses were performed using platforms that provided high-resolution scans. The results from the mass spectrometry were processed and compared with the Swiss-Prot mouse database. The researchers only selected proteins containing at least two peptides and having a false discovery rate of 0.01.

Fold changes, which is a quantitative metric based on the ratio of quantities between two groups or before and after treatment, were used to identify the differentially expressed proteins in the retinoid acid-induced cleft palate group. Statistical tests were then used to assess the significance of the identified difference in expression.

Volcano plots, which are scatter plots built to identify significant changes in data, and heatmaps were used to visualize the top 20 differentially expressed proteins.

The Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO) pathways were examined for the functional enrichment analysis of the identified differentially expressed proteins. The researchers identified the key hub proteins involved in cleft palate formation by analyzing protein-protein interaction networks.

Major Findings

The study identified 25 proteins that were upregulated and 19 proteins that were downregulated in the retinoic acid-induced cleft palate group of mice in comparison to the normal control group. These proteins were largely involved in motor protein activity, forming the actin cytoskeleton, extracellular matrix organization, and myosin complex formation.

The enrichment analysis conducted to identify the roles of the differentially expressed proteins confirmed the roles of these proteins in palate development, as most of the proteins were involved in external structure formation and the organization of the extracellular matrix.

The cellular component analysis further confirmed the involvement of these proteins in cell structure and organization based on the enrichment in myosin complex, actin cytoskeleton, and collagen-containing extracellular matrix.

The molecular function analysis also highlighted their importance in palate development based on their involvement in maintaining actin dynamics and the integrity of the extracellular matrix. The KEGG pathway analysis indicated that these differentially expressed proteins were also involved in interactions with extracellular matrix receptors and in motor protein function.

Ten key hub proteins were identified through protein-protein interaction analysis. These include motor proteins such as troponin C2 (Tnnc2), myosin light chain 1 (Myl1), myosin heavy chain 8 (Myh8), and myosin heavy chain 3 (Myh3). Additionally, extracellular matrix proteins were identified, including collagen type II alpha 1 chain (Col2a1), collagen type IX alpha 1 chain (Col9a1), matrilin 3 (Matn3), and aggrecan (Acan). Creatine kinase (Ckm) and myosin light chain 4 (Myl4) were also part of the key hub proteins.

Conclusions

To summarize, the study provided insights into the molecular mechanisms of cleft palate development and highlighted the roles of extracellular matrix and motor proteins.

The differential expression of the ten hub proteins identified in this study could potentially serve as biomarkers for effective prenatal screening and potential therapeutic approaches. Further studies using knock-out experiments could confirm the involvement of these proteins in cleft palate formation.