Protein kinase A (PKA), also known as a cyclic adenosine monophosphate (cAMP)-dependent protein kinase, is a prototypical kinase that exists as a tetrameric holoenzyme in normal conditions (9). It comprises a dimer of regulatory (R) subunits and two catalytic (C) subunits. PKA plays a pivotal role in integrating upstream second messenger signals, such as cAMP, with each step tightly regulated to ensure proper homeostatic signaling. However, mutations in specific genes can disrupt this signaling process, leading to a range of diseases (10).

Gsα, the predominant transcript of Gnas, primarily participates in the PTH/PTH-related protein (PTHrP)-cAMP-PKA signaling pathway (11). The PTH-activated signal transduction cascade involves G protein-coupled receptors (GPCRs) and numerous downstream intracellular effectors, including heterotrimeric stimulatory G proteins (Gs), cAMP-dependent PKA, and cAMP-specific phosphodiesterases (PDEs). Upon binding of PTH and PTH-related peptide (PTHrP) to the PTH1 receptor (PTH1R), the activation of Gsα stimulates the generation of the intracellular cAMP, thus initiating the signaling pathway (12–14).

In proximal renal tubules, the production of cAMP leads to a decrease in phosphate levels and an increase in vitamin D levels, which is essential for maintaining normal calcium balance. This mechanism could explain the hyperphosphatemia and hypocalcemia observed in patients with pseudohypoparathyroidism caused by inactivating mutations of Gsα. Conversely, in patients with paternally inherited pseudopseudohypoparathyroidism (PPHP), their kidneys do not exhibit resistance to PTH, consistent with the preferential expression of Gsα maternal alleles in proximal renal tubules, thyroid, ovaries, and pituitary (15). These conditions will be further described in the subsequent sections.

Similar to PTH, PTHrP primarily signals via cAMP. In bone tissues, PTHrP’s interaction with PTH1R is essential for maintaining chondrocyte proliferation during bone development. The production of cAMP activates PKA and inhibits salt-inducible kinase (SIK), subsequently regulating myocyte enhancer factor (MEF2c), ultimately leading to chondrocyte proliferation (16, 17) ( Figure 2 ).

Additionally, Gsα can also influence bone development through multiple signaling pathways, such as Wnt/β-catenin and Hh signaling pathways. These pathways are essential for determining the fate of osteoblasts and can also regulate bone mass after bone formation (18, 19). It has been reported that Gsα signaling regulates skeletal development and homeostasis through the Wnt/β-catenin pathway (20). In Gsα ^m+/p- mice, cortical bone shows elevated expression of the Wnt inhibitors such as sclerostin and Sfrp4, consistent with a decrease in Wnt/β-catenin signaling. Gsα enhances Wnt signaling, and thus promotes the differentiation of mesenchymal progenitor cells into osteoblast lineages (21). Enhanced Wnt-β-catenin signaling pathway has also been observed in bone tissue from FD patients. In these patients, bone marrow mesenchymal stem cells exhibit increased bone progenitor markers and reduced mature osteoblast markers. While activation of Gsα protein alone was not sufficient to activate Wnt/β-catenin pathway, it can enhance Wnt/β-catenin signaling activity by facilitating its association with Lrp5/6 through binding to Axin (22). Thus, Gsα regulates bone formation through at least two distinct mechanisms: promoting mesenchymal progenitor cells to enter the osteoblastic lineage, while enhancing Wnt signaling and inhibiting the differentiation of osteoblasts that have already embarked on the osteoblastic lineage (23).

Furthermore, Gsα signaling acts as an inhibitor of the Hh signaling pathway. Animal studies have demonstrated that the absence of Gsα leads to the upregulation of the Hh signaling pathway. Mechanistically, Gsα inhibits Hh signal transduction by generating cAMP and activating PKA (20, 24).

Both Wnt/β-catenin and Hh signaling pathways act downstream of the Gsα signaling pathway, with Gsα serving as a critical regulator by maintaining a delicate balance between these pathways for proper osteoblast differentiation ( Figure 3 ).

At the biochemical level, XLαs shares functional similarities with Gsα by promoting the generation of cAMP upon activation of GPCRs (25, 26). On the other hand, researchers have elucidated the role of XLαs in mediating PTH signaling.

XLαs plays a crucial role in mediating the renal effects of PTH by enhancing Gq/11 signaling, promoting the production of inositol trisphosphate(IP3), and activating specific isoforms of protein kinase C (PKC). This intricate cascade ultimately regulates the homeostasis of phosphate and vitamin D during early postnatal development (27).

Subsequent studies have revealed a novel mechanism involving XLαs/IP3/PKC pathway in the synthesis of Fibroblast Growth Factor 23(FGF23), a hormone originating from bones and bone marrow that play a crucial role in maintaining phosphate balance. FGF23 acts on the kidneys, reducing the reabsorption of phosphate in the proximal tubules and inhibiting the synthesis of 1,25(OH)2D (28).

The researchers observed that the deletion of XLαs in Ocy454 cells, which resemble osteocytes, resulted in reduced FGF23 mRNA levels, diminished IP3, and decreased PKCα/PKCδ protein levels. In mice, FGF23 production was induced by injecting phorbol myristate acetate (PMA) or by activating Gqα-Q209L in osteocytes and osteoblasts, dependent on the MAPK signaling pathway ( Figure 4 ) (29).

Recent advancements have uncovered an intriguing mechanism underlying the activation of phosphoinositide turnover by Gs-coupled receptors. Specifically, investigations have demonstrated that a distinct isoform of phospholipase Cβ (PLCβ), namely PLCβ4, exhibits specific responsiveness to stimulation in cells transfected with XLαs and purified protein components. Notably, PLCβ4 is essential for facilitating the accumulation of phosphatidylinositol in Ocy454 cells following isoproterenol-induced signaling (30).

Fibrous dysplasia (FD) is a rare and complex skeletal disorder characterized by the replacement of bone and marrow by fibrous tissue, resulting in fragile bones (31).The symptoms of FD can involve single or multiple bones, and monostotic fibrous dysplasia is more common than polyostotic, usually involving the femur or craniofacial bones (32–34). In some cases, FD can damage the maxilla or mandible, further affecting the dental development (35, 36). Key clinical features include bone deformities and fractures, which may present various functional impairments, such as vision, hearing, and walking difficulties in severe cases (37, 38).

McCune-Albright syndrome (FD/MAS) is the combination of FD with various extraosseous features. Commonly affected areas include the skin (characterized by hyperpigmented macules known as cafe-au-lait skin spots), a variety of endocrine tissues (resulting in precocious puberty, hyperthyroidism, or growth hormone overload), and occasionally skeletal muscle (intramuscular mucinous tumors, also known as Mazabraud syndrome) (39). FD/MAS exhibits mosaic patterns at the tissue level, with both mutation-positive and mutation-negative osteoblasts present. The presentation varies among patients because of the wide distribution of Gsα and the mosaic nature of FD (40). FD/MAS arises from somatic and functionally acquired mutations in GNAS, typically occurring in exon 8 and causes heterozygous missense mutations at R201 position, converting arginine 201 to histidine (R201H) or cysteine (R201C) (41, 42). Occasionally, arginine can also be replaced by serine, glycine, or leucine (43). Different codons may also be affected, for instance, codon 227 (in exon 9; Q227), which can lead to FD (44). Mouse and in vitro models based on these mutations disrupt Gsα’s intrinsic GTPase activity during skeletal development, leading to constitutive activation and excessive cAMP production (45). Constitutive Gsα signaling impairs the differentiation of mutant osteoprogenitor cells, as observed in in vitro studies of cells isolated from FD lesions and normal human osteoprogenitor cells stably transduced with Gsα ^R201C (23, 46). Proliferation of undifferentiated skeletal stem cells produces fibrous bone tissue, accompanied by the loss of normal bone marrow functions like hematopoiesis. Thus, FD is characterized by a lack of hematopoietic cells and adipocytes in the bone marrow, which is replace by fibrotic tissue. Abnormal bone structure, mineral content, and trabecular manifestations stem are attributed to aberrant osteoblast activity, potentially through Wnt/β-catenin signaling. Osteoclast production may also be promoted locally by osteoclast-promoting factors, such as interleukin-6 and receptor activator of nuclear kappa-B ligand. Additionally, inherent mineralization defects and phosphorylated hormone fibroblast growth factor-23 (FGF23) produced by FD injury contribute to severe osteomalacic changes (20–23, 47).

Albright’s Hereditary Osteodystrophy (AHO) is a condition caused by mutations in the GNAS gene, characterized by various physical traits, including short stature, brachydactyly, obesity, a round face, cognitive impairment, and the abnormal formation of bone outside the skeletal system, referred to as heterotopic ossification (48, 49). These features were first described by Albright et al., hence the name Albright’s hereditary osteodystrophy (50, 51).

Heterotopic ossification is the most devastating feature of AHO. It involves the formation of bone in soft tissues, primarily through intramembranous ossification (52–54). Ectopic ossification in patients with AHO occur within the dermis and subcutaneous tissue, causing painful lesions that may require surgical intervention. These lesions can appear spontaneously or be triggered by various factors from minor trauma to major surgery or extensive wounds (55). Histological analysis shows the presence of mineralized bone tissue with a bone marrow component in the biopsy specimen, while no cartilage is detected, indicating an origin primarily in intramembranous bone formation. Importantly, these ectopic ossifications can occur regardless of whether the Gsα mutation is inherited from the mother or father, reflecting Gsα haploinsufficiency. Notably, they are not associated with abnormal levels of calcium and phosphate (56, 57). Gsα plays a vital role in ectopic ossification, since reduced Gsα levels in human mesenchymal stem cells lead to increased bone formation and inhibited adipocyte formation (20, 58). Removing Gsα from progenitor cells expressing osterix reduces the number of bone progenitor cells while enhancing osteoblast differentiation. Additionally, Runx2, a gene marker for osteoblasts, seems to inhibit the expression of Gsα, suggesting an opposing role of Gsα in osteogenic differentiation (59).

Pseudohypoparathyroidism (PHP) constitutes a rare group of endocrine disorders, initially reported by Albright and colleagues in 1942, characterized by complete or partial failure of end-organ to respond to PTH. PHP is manifested by variable degrees of hypocalcemia and hyperphosphatemia (51). The exact global prevalence of PHP is uncertain, but is estimated to be around 0.79 cases per 100,000 people, with a slightly higher prevalence of 1.2 cases per 100,000 in Japan (60, 61).

PHP is classified according to the level of urinary cAMP after bovine parathyroid extract injection. PHP type I (PHP I) differs from PHP type II (PHP II) by the abnormal urinary cAMP response to exogenous PTH stimulation. Within PHP I, there are three subtypes (Ia, Ib, and Ic), depending on the specific defects in the GNAS gene. These defects can result from heterozygous inactivating mutations or imprinting defects on the maternal allele. When heterozygous inactivating mutations are in the paternal allele, the mutations lead to pseudo-pseudohypoparathyroidism (PPHP) and progressive osseous heteroplasia (POH). PHP Ia/PHP Ic and PHP Ib are distinguished by the presence or absence of AHO. Hormonal resistance helps distinguish PHP I from PPHP. Additionally, an in vitro assay that measures Gsa protein activity from erythrocyte membranes can differentiate between PHP Ia and PHP Ic, as Gsα activity is reduced in PHP Ia but normal in PHP Ic, although this may be attributed to the assay conditions rather than true biological differences (61, 62). These PHP subtypes are typically caused by genetic mutations inherited in an autosomal dominant manner, or by epigenetic, sporadic, or genetic changes within or upstream of the GNAS gene ( Table 1 ).

The previous classification of PHP did not consider the molecular defects, omitting conditions like acrodysostosis, POH, and PTH1R-related chondrodysplasia (63). A new classification system has been established by the EuroPHP network, encompassing all disorders related to inactivation of the PTH/PTH1R signaling pathway (iPPSD). This nomenclature employs numerical identifiers to characterize specific subtypes, enabling the description of both clinical and molecular features. For instance, iPPSD1 represents a loss-of-function variant in PTHR1, iPPSD2 corresponds to a loss-of-function alteration in GNAS, iPPSD3 denotes methylation defects at one or more GNAS DMRs, iPPSD4 signifies a pathogenic variant in PRKAR1A, iPPSD5 indicates a pathogenic variant in PDE4D, iPPSD6 relates to a pathogenic variant in PDE3A, and iPPSDx is used when no molecular defect is identified. These classifications focus on categorizing conditions based on their underlying mechanisms and can aid in guiding diagnosis through major and minor diagnostic criteria (64, 65).

Patients affected by PPHP show physical characteristics of AHO but do not experience hormone resistance (97). It is characterized by normal PTH, calcium, and phosphorus levels (98). The pathogenesis involves a heterozygous loss-of-function mutation in the paternal GNAS gene, resulting in decreased Gsa activity in vitro. PPHP Patients may exhibit short stature, brachydactyly but generally do not have mental retardation or obesity. These patients tend to be smaller by gestational age and have lower birth weights compared to those with PHP Ia, and patients carrying exon 2-13 mutations were even smaller at birth than those carrying exon 1 mutations, suggesting that the paternally expressed GNAS proteins, including XLαs is involved in fetal growth (99, 100).

POH represents another disorder in which patients often develop cutaneous ossifications early in life, involving deep connective tissue and skeletal muscle (101). Ectopic ossification of POH is formed mainly through intramembranous osteogenesis. However, a histopathological examination of subcutaneous ossification in one patient revealed the presence of chondrocyte clusters, a feature found in fibrous developmental abnormalities. Most lesions in patients begin in infancy with cutaneous or subcutaneous ossification and subsequently become invasive, and patients often present with severe ankylosis of the affected joints, which limits growth. In most patients with POH, heterotopic bone formation appears to be an isolated clinical manifestation with no other typical AHO features, while most GNAS mutations causing POH are paternally inherited (102–104).

Mutations in paternal GNAS exons 2-13, and less frequently exon 1, cause this severe form. This also leads to the hypothesis that POH is primarily related to a deficiency of the paternal XLαs protein rather than a deficiency of the paternal Gsα. Recently, Jing Ma et al. identified a de novo mutation (c.175C > T, p.Q59X) in exon 2 of the GNAS gene in a clinical case of POH (105–107).

Akio Sakamoto et al. used Gsα-floxed mice (128) with collagen Iα1 promoter Cre recombinase transgenic mice (129) to create osteoblast/osteocyte-specific Gsα deficient (BGsKO) mice. These mice exhibited similarities to PTH deficient mice (130). In neonatal BGsKO mice, craniofacial skeletal dysplasia was observed, with thickening of the mandible, maxilla, and zygomatic arch, especially Merkel’s cartilage, which had ossified in neonatal BGsKO mice. Long bones in BGsKO mice displayed defective primary spongiosa formation, immature osteoid (new bone formation), and reduced total length, leading to decreased trabecular volume. In contrast, cortical bone thickened with a narrowed bone marrow cavity, possibly due to reduced cortical bone resorption caused by a decrease in osteoclasts on the cortical bone surface. Altered expression of alkaline phosphatase and reduced expression of bone-bridging protein and osteocalcin suggested a decrease in mature osteoblasts in the bone. The expression of osteoclast-stimulating factor receptor activator of NF-κB ligand (RANKL) was also reduced (131).

Zhang et al. investigated transgenic mice with osteoblastic Gsα overexpression (HOM-Gs mice) driven by the 3.6-kilobase (kb) Col1A1 promoter. Both male and female HOM-Gs mice displayed elevated bone turnover, overactive osteoblasts, and osteoclasts, culminating in a high bone mass phenotype with notably diminished bone mass. At the cellular level, these alterations were brought about by increased bone resorption by osteoclasts and significantly enhanced bone formation by osteoblasts. Their findings indicate that elevated Gsα expression contributes to skeletal abnormalities, wherein bone production occurs at the expense of bone mass (132).

Joy Y. Wu et al. generated GsαOsxKO mice by deleting Gsα from cells expressing Osterix to study its role in the early osteoblast lineage. Such mice developed severe osteoporosis and fractures at birth and did not survive to weaning period. This phenotype results from impaired bone formation rather than increased bone resorption. Osteogenic differentiation is accelerated, and osteoblasts rapidly differentiate into osteocytes, producing woven bone. Also, the number of committed osteoblast progenitors has reduced in bone marrow stromal cells, and cranial osteocytes in cKO mice and osteoblasts are reduced. Wnt signaling was reduced in the osteoblast lineage, and expression of the Wnt signaling inhibitors sclerostin and dickkopf1 (Dkk1) was significantly increased. Their study suggests that Gsα signaling is essential for early normal bone formation (23).

Fulzele et al. generated mice lacking Gsα in osteocytes by crossing Dmp1-Cre mice with Gsα ^flox/flox mice. OCY-GsαKO animals have low bone mass and decreased adipose tissue and develop a myeloproliferative phenotype. In these mice, they investigated the impact of Gsα deficiency on the bone marrow microenvironment. The deficiency of Gsα in osteocytes led to an increase in bone marrow, spleen, and peripheral blood, resulting from alterations in the bone marrow microenvironment in mice. This effect was independent of osteoblasts or the Wnt-βcatenin pathway. The authors demonstrated that osteocytes could regulate myelopoiesis through a Gsα-dependent mechanism by secreting multiple factors, including granulocyte colony-stimulating factor (G-CSF) (133).

Subsequently, the authors’ team further studied such mice and found that the cortical bone parameters were significantly reduced in all 7-week-old Dmp1KO mice by microCT, with no change in distal femoral trabecular bone volume in females but a significant reduction in distal femoral trabecular bone volume in males. Bone trabeculae were significantly reduced in cKO mice by static histomorphometric analysis. Assays of viable osteoblasts showed a dramatic decrease in the number of osteoblasts per bone perimeter. Dynamic histomorphometric analysis showed a significant decrease in the rate of bone formation and mineral attachment per bone surface in cKO mice. Serum levels of osteocalcin and procollagen type I amino-terminal propeptide PINP were significantly reduced in cKO mice. Differently, the number of osteoclasts per total area was significantly reduced in female mice, whereas no change was observed in the male group. Combined with the authors’ previous study: the deletion of Gsα in osteocytes leads to sclerostin expression in vitro and in vivo (134). Immunohistochemistry also showed a significant increase in sclerostin expression in osteoblasts of Dmp1-KO mice. PTH can inhibit sclerostin gene expression through the Gsα signaling pathway (135, 136). Treatment of these animals with sclerostin-neutralizing antibodies rescued the number and size of osteoblasts in the cortex as well as partially rescued the low bone mass phenotype. These data suggest that a deficiency of Gsα in osteoblasts can lead to osteoporosis, in part due to an increase in sclerostin (137).

Regard et al. used Prx1-Cre (138) mice mated with Gsα ^flox/flox (139) mice to produce Prx1-Cre; Gsα ^f/- and Prx1-cre; Gsα ^f/f mice, which had a similar phenotype: born with webbing between fingers, joint fusion and progressive heterotopic appearance in the soft tissues ossification Extraskeletal mineralization was first detected at days E16.5 and E17.5, accelerated during the perinatal period, and became extensive four days after birth, with mineralization in the interphalangeal area and between the ulna and radius, leading to osseous fusion at P4. At P20, most mutant mice die due to extensive bone and joint fusion and mineralization of tendons. The authors also used Dermo1-Cre (mesoderm-derived bone and muscle tissue) or Ap2-Cre (more extensive than the former) to remove Gsα, again with a similar phenotype of ectopic ossification, so that Gnas inhibits ectopic ossification in multiple mesenchymal tissues. The authors also performed adenoviral subcutaneous injections of Ad-Cre or Ad-GFP on 4-week-old Gnas ^flox/flox mice. They found ectopic osteoblasts and mineralization in dermal and subcutaneous areas six weeks after Ad-cre injection, along with extensive ectopic ossification detected. The longer the time, the more severe this induction produced ectopic ossification and invasion of deep muscle tissue. It is shown that loss of Gnas in adult subcutaneous mesenchymal tissue is sufficient to cause ectopic ossification similar to that in POH and AHO. In vitro, they isolated bone marrow stromal cells (BMSC) and subcutaneous mesenchymal cells (SMP) from Gnas^flox/flox mice, which showed accelerated osteogenic differentiation as well as stronger expression of osteogenic differentiation markers after removal of Gsα with Ad-Cre infection. They also improved the phenotype of ectopic mineralization by removing Gli2 (relaying Hh signaling) in mutant mice and found that its removal improved or even rescued ectopic ossification, as did injection of ATO or GANT58, pharmacological inhibitors of Gli, into female mice harboring Prx1-Cre; Gsα ^f/- pups. Inhibition of osteoblast differentiation by ANT-58 was also observed in Gsα-deficient BMSCs was observed in vitro. In conclusion, their study demonstrated the ability of Gsα to confine bone formation within the skeleton by inhibiting Hh signaling in mesenchymal progenitor cells. In conjunction with their previous studies (22), the researchers determined that Gsα serves as a critical regulator of osteoblast differentiation by maintaining the balance between two essential signaling pathways: Wnt/β-catenin and Hh (24).

Xu et al. removed Gsα from osteochondral progenitor cells by generating Prx1-Cre; Gsα ^flox/flox mice, resulting in accelerated cranial bone formation during craniogenesis and cranial malformation after birth. This is because the loss of Gsα activated the Hh signaling pathway and accelerated osteoblast differentiation and ossification during cranial bone development, but the bone formed was of low quality and low mineral density, which may be due to increased osteoclast differentiation (140).

The premature closure of the cranial suture caused by GNAS deficiency was further investigated by Xu et al. The authors found inactivation of the Hh signaling pathway in single-cell sequencing of cranial suture chondrocytes from normal neonatal mice, which, consistent with the previous mention, was associated with the activation of Gsα. The authors deleted Gsα in cranial chondroprogenitor cells by crossing Gsα ^flox/flox with Prx1 -Cre mice. These mice developed severe cranial and limb bone phenotypes, with most mutant mice dying at P6. Prx1-Cre; Gsα ^flox/flox mice developed premature closure of the cranial suture, similar to the cranial malformation of mutant patients (141). Prx1-Cre; Gnas ^fl/fl mice at postnatal day 14 showed ectopic ossification in the sagittal suture, posterior fontanel, lambdoid suture, and mastoid fontanel. Subsequent histological analysis revealed that the earliest cranial suture phenotype could be observed in E16.5 sections. At P0, the mineralization of cranial cartilage in mice occupied half of the cartilage area, and the mutant cells were hypertrophic and mineralized compared with chondrocytes in normal controls. Next, the authors mated R26RtdTom reporter mice with Prx1-Cre; Gsα ^flox/flox mice. Immunostaining revealed increased expression of ColX, Osx, and OPN in the mutant chondrocytes, while the mutant chondrocytes did not express osteogenic markers. This suggests that the deletion of Gsα within chondrocytes allows them to transform into an osteoblast lineage through hypertrophy. They also used Sox9-CreER and Osx-Cre mice to study the function of Gsα in chondrocytes and the effect of mutated osteoblasts on chondrocytes, respectively. Sox9-CreER; Gnas ^fl/fl mice reproduced ectopic mineralization in mastoid fontanel after being induced at P6. No chondrogenic mineralization or premature fusion of the cranial suture and fontanel was found in the cranial vault of Osx-Cre; Gnas ^fl/fl mice at P0 compared to control Osx-Cre mice; therefore, the authors concluded that premature closure of the cranial suture was the result of ectopic ossification of cranial suture mesenchymal cells and chondrocytes. Next, the authors crossed mice with the Ptch1LacZ allele with Prx1-Cre; Gsα ^flox/flox mice. By X-gal staining, t they found that the cranial suture and fontanelle of the mutant mice had substantial activation of Hh signaling. Also, coronal sections of mammillary fontanelles from Prx1-Cre; Gnas^fl/fl; Ptch1LacZ mice had large numbers of LacZ⁺ chondrocytes, while controls had no LacZ⁺ chondrocytes. These data suggest that the regulation of chondrocyte fate by Gsα is associated with the activation of Hh signaling. Reduction of Gli transcription activity by crossing with a loss-of-function Gli2 allele or injecting GLI1/2 antagonist hindered the progression of cartilage HO in neonatal stage mice (142). This implies that reducing Hh signaling could be an intervention to suppress the cranial deformity caused by Gnas deficiency (140).