The Sphingosine-1-Phosphate/RhoA/Rho-Associated Kinases/Myosin Light Chain Pathway in Detrusor of Female Rats Is Down-Regulated in Response to Ovariectomy
Perimenopausal women often experience genitourinary syndrome of menopause (GSM), characterized by dysuria, urinary frequency, and incontinence. These symptoms are associated with reduced detrusor contractility, though the underlying mechanisms remain poorly understood. Emerging evidence suggests that the sphingosine-1-phosphate (S1P) pathway, which regulates smooth muscle contraction and is influenced by estrogen, may play a role in this process. A recent study utilizing an ovariectomized (OVX) rat model explored how estrogen deficiency impacts the S1P/RhoA/Rho-associated kinases (ROCK)/myosin light chain (MLC) pathway in detrusor tissues, revealing critical insights into the molecular basis of dysuria during menopause.
Estrogen Deficiency and Dysuria in Menopause
Menopause results from the decline of ovarian hormone production, leading to estrogen deficiency. Urinary symptoms such as dysuria are common in GSM, often linked to detrusor dysfunction. While estrogen replacement therapy alleviates some symptoms, the specific pathways through which estrogen modulates detrusor contraction are not fully defined. The S1P pathway, which regulates smooth muscle tone via RhoA/ROCK/MLC signaling, has been implicated in bladder function. S1P, a bioactive sphingolipid, binds to G protein-coupled receptors (S1PR1/2/3), activating RhoA and ROCK. This cascade enhances phosphorylation of MLC (p-MLC), a key determinant of smooth muscle contraction. Studies suggest that estrogen modulates S1P levels, as premenopausal women exhibit higher plasma S1P than postmenopausal women or men.
Experimental Design and Model Validation
To investigate how estrogen deficiency affects the S1P/RhoA/ROCK/MLC pathway, 36 female Sprague-Dawley rats were divided into three groups: sham-operated (SHAM), ovariectomized (OVX), and ovariectomized with estrogen supplementation (E). Successful ovariectomy was confirmed via vaginal cytology and radioimmunoassay. The OVX group showed a significant reduction in serum estrogen (5.24 ± 1.44 pg/mL vs. SHAM: 32.02 ± 3.14 pg/mL, P < 0.05), which was restored in the E group (32.69 ± 3.12 pg/mL). Detrusor tissues were analyzed using quantitative PCR (qPCR), Western blotting (WB), and enzyme-linked immunosorbent assay (ELISA) to evaluate mRNA, protein, and S1P levels.
Downregulation of S1P Synthesis and Signaling
The S1P pathway was significantly impaired in OVX rats. Sphingosine kinase 1 (SphK1), the enzyme responsible for S1P production, showed a 39% reduction in mRNA and a 45% decrease in protein expression (P < 0.05). SphK2, another S1P-synthesizing enzyme, remained unchanged. Correspondingly, S1P content in detrusor tissues was 18.86 ± 0.69 nmol/g in OVX rats, a 13% reduction compared to SHAM (21.73 ± 1.09 nmol/g, P < 0.05). Estrogen supplementation normalized SphK1 expression and S1P levels (E group: 21.85 ± 0.58 nmol/g).
At the receptor level, S1PR2 and S1PR3 mRNA levels in OVX rats decreased by 25% and 27%, respectively (P < 0.05), while S1PR1 was unaffected. However, protein levels of S1PR2/3 remained unchanged, suggesting post-transcriptional compensation or delayed protein turnover. This discrepancy highlights the complexity of receptor regulation under short-term estrogen deprivation.
Impairment of RhoA/ROCK/MLC Contractile Signaling
Downstream of S1P, the RhoA/ROCK/MLC pathway was also disrupted. ROCK2, the primary isoform regulating smooth muscle contraction, exhibited a 36% reduction in mRNA and a 41% decrease in protein expression (P < 0.05). Although RhoA, ROCK1, MYPT1, and MLC20 mRNA/protein levels remained stable, phosphorylation of key regulatory sites plummeted. Phosphorylated MYPT1 (p-MYPT1) and MLC20 (p-MLC20) levels dropped by 54% and 47%, respectively (P < 0.05). These changes indicate suppressed ROCK activity, which normally inhibits MLCP to sustain p-MLC levels and muscle contraction. Estrogen treatment restored ROCK2 expression and phosphorylation of MYPT1/MLC20 to baseline levels.
Mechanistic Insights and Clinical Implications
The study demonstrates that ovariectomy-induced estrogen deficiency downregulates the S1P/RhoA/ROCK/MLC pathway in the detrusor. SphK1 appears to be the primary S1P-producing enzyme influenced by estrogen, as SphK2 remained unaffected. This aligns with prior studies showing estrogen-dependent SphK1 activation in vascular and breast tissues. The reduction in S1PR2/3 mRNA but stable protein levels suggests a potential compensatory mechanism preserving receptor function temporarily. The selective decline in ROCK2, rather than ROCK1, underscores its dominant role in contraction.
The findings align with clinical observations of detrusor hypofunction in postmenopausal women. Reduced S1P and ROCK2 activity likely diminish MLC phosphorylation, impairing actin-myosin cross-bridge formation. Estrogen’s restorative effects highlight its therapeutic potential, though the exact mechanism linking estrogen to SphK1 activation remains unclear. Previous work implicates membrane estrogen receptors (e.g., GPR30) in SphK1 regulation, suggesting non-genomic signaling pathways.
Limitations and Future Directions
While the study establishes a clear link between estrogen deficiency and S1P pathway suppression, several questions remain. The lack of S1PR2/3 protein changes despite mRNA reductions may reflect assay sensitivity or temporal dynamics. Longer-term studies could clarify whether receptor protein levels eventually decline. Additionally, functional assays measuring detrusor contractility would strengthen the correlation between molecular changes and physiological outcomes.
Future research should explore the upstream signaling mechanisms, such as how estrogen receptors interact with SphK1 and whether S1P receptor agonists can rescue detrusor function in OVX models. Investigating crosstalk between S1P and other estrogen-regulated pathways, such as nitric oxide signaling, may further elucidate menopausal bladder dysfunction.
Conclusion
This study provides compelling evidence that ovariectomy disrupts the S1P/RhoA/ROCK/MLC pathway in the detrusor, primarily through estrogen-dependent regulation of SphK1 and ROCK2. These molecular changes likely contribute to the urinary symptoms observed in perimenopausal women. Restoring estrogen levels normalizes the pathway, underscoring its potential as a therapeutic target. By unraveling the mechanistic basis of detrusor hypofunction in menopause, this work paves the way for novel treatments targeting S1P signaling to improve quality of life in GSM patients.
doi.org/10.1097/CM9.0000000000000767
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