Extracellular protein kinase A and G are potential biomarkers of some inflammation-associated disorders

Introduction

Inflammation-associated disorders are characterized by dysregulation of immune response, including a group of common, chronic, and complex disorders, such as type-1 diabetes, hypertension, coronary heart disease, rheumatoid arthritis, inflammatory bowel disease, autoimmunee diseases. Together, inflammation-associated disorders have an estimated incidence of 80 per 100 000 person-years and affect 3–5% of the population.^1,2 Chronic diseases result in significant health care and high diseases burden. Protein kinases, especially, cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), ³ as signal cascades, play key roles in inflammation-associated disorders.^3,4 Recently, more and more data indicated the secretion phenomenon of PKACα and used a biomarker for tumor diagnosis.^5–7 Our group also noticed that PKACα and PKG II could be detected in the serum of human and mice, as well as in the cell culture supernatant.^8,9 Since PKACα and PKG II play an important role in inflammation-associated disorders, it remains unclear whether the secreted PKACα and PKG II can serve as potential biomarkers for these diseases. Therefore, in the present study, we assess the diagnostic effects of PKACα and PKG II for some of inflammation-associated disorders, such as, coronary disease, diabetes, rheumatoid arthritis, and schizophrenia. Our results showed that the PKACα and PKG II in the serum could be used for screening, diagnosis of coronary disease, rheumatoid arthritis, and schizophrenia.

Materials and Methods

Results

The levels of PKACα and PKG II were significantly changed in patients

To clarify the differences in patients suffering from coronary disease, rheumatoid arthritis, schizophrenia and depression, and volunteers, the serum PKACα and PKG II levels were analyzed. The results showed that PKACα was significantly decreased in the serum of coronary disease, rheumatoid arthritis, and schizophrenia, compared to volunteers (*p < .05, *p < .05, ** p < .01, respectively). Conversely, PKG II was increased in the serum of rheumatoid arthritis, schizophrenia, and depression disorders compared to their volunteers (**p < .01, ***p < .001, *p < .05, respectively). There was no difference in PKACα in patients with diabetes and depression disorders compared to corresponding volunteers (p > .05). Besides, for PKG II, there were no obvious changes between diabetes and volunteers, as well as coronary disease and volunteers (p > .05) (Figure 1(a) and (b)).

OPEN IN VIEWER

Figure 1.

The levels of PKACα and PKG II in the serum of different patients with inflammation-associated disorders. (a) The level of PKACα in human sera from Control healthy subjects (n = 20, 131.4 ± 37.49), patients with Diabetes (n = 42, 118.9 ± 23.45) or Coronary disease (n = 31, 111.9 ± 21.34), Rheumatoid arthritis (n = 18, 107.4 ± 8.466), Schizophrenia (n = 30, 104.6 ± 15.81), and Depression disorder (n = 30, 130.1 ± 37.21) were analyzed by the ELISA arrays. (b) The level of PKG II in human sera from Control healthy subjects (n = 20, 121.3 ± 55.79), patients with Diabetes (n = 42, 100.5 ± 21.24) or Coronary disease (n = 31, 124.2 ± 42.76), Rheumatoid arthritis (n = 18, 173.1 ± 22.19), Schizophrenia (n = 30, 171.0 ± 67.48), and Depression disorder (n = 30, 154.8 ± 32.63) were analyzed by the ELISA arrays. The levels represent the mean ± Std. Deviation (M ± SD) of PKACα or PKG II level. *p < .05, ** p < .01, ***p < .001, versus Control; ns, not significant. NC group, normal saline-treated group.

The correlation analysis of serum PKACα and PKG II with various biomarkers in some inflammation-associated disorders

The Pearson correlation analysis was used to evaluate the relationship between serum PKACα/PKG II with varous biochemical biomarkers in different inflammation-associated disorders. In diabetes, there was a significant positive correlation between creatinine and PKACα (r = 0.3996, p = .0478), creatinine and PKG II (r = 0.4417, p = .0271), while no obvious relationship between PKACα or PKG II with glucose, glycosylated hemoglobin (HBA1C) %, and urea nitrogen. For coronary disease, there was a significant negative correlation between cholesterol (CHOL) and PKACα (r = −0.3907, p = .0328), triglyceride (TG), and PKACα (r = −0.3951, p = .0307). In rheumatoid arthritis, PKACα and Ig, RF, CRP, ESR were negatively correlation (r = −0.6193, p = .008; r = −0.3698, p > .05; r = −0.5064, p > .05; r = −0.3149, p > .05), while PKG II and Ig, RF, CRP, ESR were positively correlation (r = 0.6554, p = .0328; r = 0.507, p > .05; r = 0.6281, p > .05; r = 0.3645, p > .05). However, there is an opposite trends of PKACα and PKG II in rheumatoid arthritis (Figure 2 and Tables 1, 2, and 3)

OPEN IN VIEWER

Figure 2.

The Pearson correlation analysis of serum PKACα and PKG II with indicators in different inflammation-associated disorders. (a) Correlation plot between PKACα or PKG II with Glucose, HBA1C%, Creatinine, and Urea nitrogen in diabetes. (b) Correlation plot between PKACα or PKG II with CK-MB, CHOL, and triglyceride in Coronary Disease. (c) Correlation plot between PKACα or PKG II with CRP, RF, Ig, and ESR in Rheumatoid arthritis. HBA1C, glycosylated hemoglobin; CK-MB, creatinine kinase, MB isoenzyme; CHOL, cholesterin; TG, triglyceride; CRP, C reactive protein; RF, rheumatoid factors; Ig, immunoglobulin; ESR, erythrocyte sedimentation rate.

OPEN IN VIEWER

Table 1.

The Pearson correlation coefficient of serum PKACα and PKG II with indicators in Diabetes.

Pearson	Glucose vs.		HBA1C% vs.		Urea nitrogen vs.		Creatinine vs.
	PKACα	PKG II	PKACα	PKG II	PKACα	PKG II	PKACα	PKG II
r	0.2881	0.1105	0.318	0.09599	0.3198	0.1016	0.3996	0.4417
p value	.1626	.5989	.1214	.6481	.1191	.6289	.0478	.0271
95% CI	−0.12 ∼ 0.61	−0.29 ∼ 0.48	−0.09 ∼ 0.63	−0.31 ∼ 0.47	−0.09 ∼ 0.63	−0.31∼ 0.48	0.005 ∼ 0.69	0.06 ∼ 0.71

CI: confidence interval. HBA1C, glycosylated hemoglobin.

OPEN IN VIEWER

Table 2.

The Pearson correlation coefficient of serum PKACα and PKG II with indicators in Coronary disease.

Pearson	CK-MB vs.		CHOL vs.		TG vs.
	PKACα	PKG II	PKACα	PKG II	PKACα	PKG II
r	−0.1678	0.03294	−0.3907	−0.04018	−0.3951	0.2984
p value	.3755	.8653	.0328	.8361	.0307	.1158
95% CI	−0.50 ∼ 0.21	−0.34 ∼ 0.40	−0.66 ∼ −0.04	−0.40 ∼ 0.33	−0.66 ∼ −0.04	−0.08 ∼ 0.60

CI: confidence interval. CK-MB, creatinine kinase, MB isoenzyme; CHOL, cholesterol; TG, triglyceride.

OPEN IN VIEWER

Table 3.

The Pearson correlation coefficient of serum PKACα and PKG II with indicators in Rheumatoid arthritis.

Pearson	RF vs.		Ig vs.		CRP vs.		ESR vs.
	PKACα	PKG II	PKACα	PKG II	PKACα	PKG II	PKACα	PKG II
r	−0.3698	0.507	−0.6193	0.6554	−0.5064	0.6281	−0.3149	0.3645
p value	.1749	.0538	.0138	.008	.0541	.0122	.2529	.1817
95% CI	−0.74 ∼ 0.18	−0.01 ∼ 0.81	−0.86 ∼ −0.16	0.22 ∼ 0.87	−0.81 ∼ 0.01	0.17 ∼ 0.86	−0.71 ∼ 0.24	−0.18 ∼ 0.74

CI: confidence interval. RF, rheumatoid factor. Ig, immunoglobulin; CRP, C-reaction protein. ESR, erythrocyte sedimentation rate.

The diagnostic potential of serum PKACα and PKG II in different inflammation-associated disorders

The optimal cutoff value of PKACα and PKG II for the different inflammation-associated disorders was selected using ROC curve analysis. As the results showed that, for coronary disease, the sensitivity and specificity of PKACα were 96.55% and 50%, respectively, the cutoff value was 134.1 pg/mL. For rheumatoid arthritis, the sensitivity and specificity of PKACα were 100% and 55% with a cutoff value of 126 pg/mL, and for PKG II was 100% and 65% with a cutoff value of 131 pg/mL. For schizophrenia, the sensitivity and specificity of PKACα were 93.1% and 60% with a cutoff value of 123 pg/mL, and for PKG II was 76.67% and 75% with a cutoff value of 137.4 pg/mL. Besides, the sensitivity and specificity of PKG II were 86.21% and 65% for depression with a cutoff value of 130.5 pg/mL. All the data indicated that PKACα and PKG II have the potential diagnostic value for the above diseases (Figure 3 and Table 4)

OPEN IN VIEWER

Figure 3.

The ROC analysis curve of PKACα and PKG II of serum in various diseases. The ROC plots were from Control healthy subjects (n = 20), patients with Coronary disease (n = 31; a), Rheumatoid arthritis (n = 18, b), Schizophrenia (n = 30, c), and Depression disorder (n = 30, d). ROC, Receiver operating characteristic.

OPEN IN VIEWER

Table 4.

The ROC analysis of serum PKACα and PKG II in various diseases.

	PKACα			PKG II
	Coronary disease	Rheumatoid arthritis	Schizophrenia	Rheumatoid arthritis	Schizophrenia	Depression
AUC	0.679	0.712	0.719	0.853	0.775	0.754
SE	0.0878	0.0938	0.0882	0.0676	0.0724	0.0771
95% CI	0.51–0.85	0.53–0.89	0.55–0.89	0.72–0.99	0.63–0.92	0.60–0.91
p Value	.0344	.0343	.0095	.0004	.0011	.0027
Youden index	0.4655	0.55	0.531	0.65	0.5167	0.5121
Sensitivity%	96.55	100	93.1	100	76.67	0.8621
Specificity%	50.00	55.00	60.00	65.00	75.00	65.00
Cutoff value	<134.1	<126	<123	>131.0	>137.4	>130.5

CI: confidence interval; AUC: Area under the ROC curve; SE: Std. Error.

Discussion

Inflammation-associated disorders include rheumatoid arthritis, inflammatory bowel disease, asthma, multiple sclerosis, connective tissue disorders, etc. Furthermore, inflammation-associated disorders are often accompanied by different co-morbidities, such as cardiovascular disease, metabolic and bone disorders, neuropsychiatric disease, and cognitive deficit, which further influences the quality of life. There are no effective measures to cure these diseases.^10–12 Therefore, it is crucial to detect and control the progression of the disease timely. However, because of the pathogenic complexity of inflammation-associated disorders, there are no broad-spectrum diagnostic markers.

PKACα and PKG II are typically considered as classical intracellular molecules and play important roles in different pathological and physiological processes. PKA contains catalytic subunits and regulatory subunits; there are three isoforms of the catalytic subunits (PKA-C: Cα, Cβ, Cγ) and four isoforms of the regulatory subunits (PKA-R: RIα, RIβ, RIIα, RIIβ). In the absence of cAMP, PKA is an inactive heterotetramer with two identical catalytic and regulatory subunits. Whereas, in the presence of cAMP, R subunits binds to cAMP, and the C subunits are released from holoenzyme, phosphorylating target substrates, ¹³ including ion channels, enzymes, and transcription factors, and regulates their activity. PKA plays multiple roles in heart functions, including contraction, metabolism, ion fluxes, gene transcription,^13,14 neuronal development, synaptic plasticity, brain homeostasis, as well as in the regulation of feeding, energy expenditure, and glucose homeostasis.^15,16 Furthermore, dysregulation of PKA signaling contributes to the progression of several neuropsychiatric and neurodegenerative diseases and inflammation-associated disorders. ¹⁷ Conversely, PKG is encoded by two genes, prkgI and prkgII, which are transcribed into three enzymes PKGIα, PKGIβ, and PKG II. The PKG I isozymes are prominent in all types of smooth muscle, platelets, and specific neuronal areas. ¹⁸ As a membrane-bound protein, PKG II is mainly located in the secretory epithelium of the small intestine, brain, kidney, chondrocytes, prostate, gallbladder, pancreas, and lung.^19–22 PKG II is composed of three functionally distinct domains ²³ : an N-terminal domain, a regulatory domain, and a catalytic domain, and the physiologically most relevant activator of PKG II is cGMP. ²¹ PKG II regulates the activity of processes in cells by phosphorylating specific target proteins on serine or threonine residues. ²¹ PKG II is known to be linked to fluid and salt balance in epithelial tissues, ²⁴ intestinal fluid secretion, ²⁵ chondrogenesis, renin secretion, and memory formation, cytoskeleton regulation, proliferation, and apoptosis in tumor cells.^26,27 However, an extracellular form of PKA and PKG II has been found in the conditioned medium and serum from cancer patients, including prostate, renal, bladder, breast, and lung carcinomas, and can be considered as a diagnosis biomarker.^6,7,28–30

The present study demonstrated that the serum PKACα and PKG II had obvious changes in coronary disease, rheumatoid arthritis, schizophrenia patients. However, serum PKACα and PKG II have opposite changes tendency, especially in rheumatoid arthritis. It seems that PKACα and PKG II can serve as potentially diagnostic markers for inflammatory associated disorders. However, our research still has some limitations, such as, the sample number not enough, the defect of comparison between the classical clinical diagnostic biomarkers and PKACα and PKG II, lack of the relationship between serum PKACα and PKG II levels and disease progression. However, our results at least indicated that the secreted PKA and PKG are potential diagnostic biomarkers for inflammation-associated disorders. Of course, to further confirm the diagnostic value of PKACα and PKG II, more clinical studies are needed.

Conclusions

Our results showed that the serum PKACα and PKG II could serve as diagnostic biomarkers for coronary disease, rheumatoid arthritis, and schizophrenia. Of course, further researches are also needed to approve the conclusions (Figure 4).

OPEN IN VIEWER

Figure 4.

The schematic image of PKACα and PKG II in different diseases. PKACα and PKG II could be secreted to the blood from the cells and regulate several immune-mediated inflammatory diseases, such as diabetes, coronary disease, rheumatoid arthritis, schizophrenia, and depression disorder. And whether extracellular PKACα and PKG II is friend or foe is also need to be investigated.