Hippo Signaling Pathway Proteins in Gingival Crevicular Fluid of Individuals With Periodontal Disease

Background and rationale

Periodontal disease is a common infectious and inflammatory condition of the periodontium, presenting clinically as gingivitis or periodontitis. Gingivitis is a reversible inflammation confined to the gingiva, whereas periodontitis is a chronic, progressive disease characterized by irreversible loss of periodontal attachment and alveolar bone. Although microbial biofilms containing periodontal pathogens initiate the disease, its severity and progression are largely governed by the host immune-inflammatory response.

The Hippo signaling pathway is a highly conserved regulatory network that integrates mechanical and biochemical cues to control cellular polarity, differentiation, proliferation, apoptosis, immune responses, tissue homeostasis, and organ size. Its core components include Yes-associated protein (YAP), transcriptional co-activator with PDZ-binding motif (TAZ), mammalian sterile 20-like kinase 1 (MST1), TEA domain family members (TEAD), large tumor suppressor kinase 1 (LATS1), and mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4). Upstream kinases such as MST1/2 and MAP4K4 activate LATS1/2, which phosphorylate YAP and TAZ, leading to their cytoplasmic retention or degradation and preventing TEAD-mediated transcriptional activity in the nucleus.

Preclinical evidence has implicated this pathway in mineralized tissue homeostasis of the alveolar bone and periodontal complex. Inhibition of YAP has been reported to suppress proliferation and induce apoptosis of periodontal ligament stem cells through the BCL-2 family pathway, and YAP/TAZ-deficient animal models show enhanced resorption of alveolar bone and cementum together with reduced alkaline phosphatase expression. Despite these findings, no study to date has directly evaluated the role of Hippo pathway proteins in human periodontal disease. This study was therefore designed to address that gap.

Objective and hypothesis

The study aimed to examine changes in the Hippo signaling pathway proteins YAP, TAZ, MST1, TEAD, LATS1, and MAP4K4, together with the osteogenic marker ALP and the anti-apoptotic protein BCL-2, in gingival crevicular fluid across periodontal health and disease states. It was hypothesized that the gingival crevicular fluid levels of Hippo pathway-related proteins are altered in periodontal disease and correlate with markers of bone turnover and apoptosis.

Study population and grouping

Systemically healthy adults aged 18 to 65 years who presented to the Department of Periodontology were assessed for eligibility. Participants were allocated into three groups of equal size according to the 2017 World Workshop on the Classification of Periodontal Diseases: periodontally healthy individuals, patients with gingivitis, and patients with stage 3 grade B periodontitis. Group assignment was based on probing depth, bleeding on probing, clinical attachment level, and radiographic evidence of bone loss.

Clinical examination

For all participants, Plaque Index, Gingival Index, Probing Depth, Bleeding on Probing, and Clinical Attachment Level were recorded by a single calibrated periodontist at six sites per tooth. Panoramic radiographs were used to assess alveolar bone loss.

Gingival crevicular fluid sampling

Gingival crevicular fluid samples were collected one day after periodontal examination. Participants were instructed not to eat, drink, or brush their teeth for at least two hours before sampling. Sampling sites were isolated with cotton rolls, supragingival plaque was removed, and tooth surfaces were gently dried. Paper strips were placed into the pocket and left in position for a fixed interval, and samples contaminated with blood or saliva were discarded. Fluid volume was measured electronically, and samples from each participant were pooled and stored at low temperature until analysis. Sampling sites within each group were defined according to group-specific clinical criteria.

Laboratory analysis

The levels of YAP, TAZ, MST1, LATS1, MAP4K4, TEAD, ALP, and BCL-2 were determined using commercially available enzyme-linked immunosorbent assay kits according to the manufacturers' instructions. Standard curves were generated individually for each biomarker, and results were expressed both as total amounts and as concentrations to account for differences in gingival crevicular fluid volume between health and disease states.

Analytical approach

Both total amounts and concentrations of the target proteins were compared among the three groups. Correlation analyses were performed to evaluate the relationships among the measured proteins and between these proteins and clinical periodontal parameters. Reporting protein levels in two complementary ways was intended to distinguish true biological differences from those attributable to changes in fluid volume associated with inflammation.

Inclusion criteria

• Systemically healthy individuals aged 18-65 years
• Non-smokers or smoking cessation more than 5 years ago
• Healthy group: No history of periodontal disease, ≥20 teeth, probing depth (PD) ≤3 mm at all sites, bleeding on probing (BOP) <10%, radiographic CEJ-to-alveolar bone crest distance ≤3 mm in ≥95% of teeth
• Gingivitis group: No history of periodontal disease, ≥20 teeth, PD ≤3 mm at all sites, BOP ≥30%, radiographic CEJ-to-alveolar bone crest distance ≤3 mm in ≥95% of teeth
• Periodontitis group: ≥15 teeth, >30% of teeth affected by periodontal disease, PD ≥6 mm, clinical attachment loss (CAL) ≥5 mm, vertical bone loss ≥3 mm, Class II or III furcation involvement, radiographic alveolar bone loss extending to the middle third or beyond (≥33%), bone loss-to-age ratio between 0.25 and 1.00

Exclusion criteria

• Periodontal treatment or antibiotic use within the past 6 months
• Fewer than 20 teeth (excluding third molars)
• Presence of any systemic disease
• Current smoking or cessation within the past 5 years
• Use of immunosuppressive medication
• Alcohol consumption
• Regular medication use
• Pregnancy or lactation
• Need for antibiotic prophylaxis prior to dental procedures
• Prosthetic restorations on the teeth selected for sampling