EIF2alpha phosphorylation can increase ATF4 so that maybe a reason to take Salubrinal in conjunction to performing LSJL as LSJL downregulates EIF2alpha phosphorylation.
Concentrated Bovine Milk Whey Active Proteins Facilitate Osteogenesis through Activation of the JNK-ATF4 Pathway., states that Bovine Milk Whey can increase ATF4 levels. Alpha-lactalbumin and beta-lactoglobulin are two proteins that make up a large portion of this milk. Lactalbumin is available for sale: Hydrolyzed Lactalbumin Protein (Pure Whey Protein) 18 oz
. I could not find lacoglobulin. However, ATF4 is a key molecule linking food intake and skeletal development., found that any high protein diet could rescue ATF4 knockout.Eukaryotic initiation factor 2 phosphorylation and translational control in metabolism.
"In response to stresses, such as nutrient deprivation and accumulation of misfolded proteins in the endoplasmic reticulum, phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α~P) reduces general translation initiation while facilitating the preferential translation of transcripts that [encode] activating transcription factor 4 (ATF4), a transcriptional activator of genes subject to the integrated stress response (ISR)."
Hedgehog proteins stimulate chondrogenic cell differentiation and cartilage formation.
"Implantation of dermal fibroblasts expressing hedgehog proteins into nude mice[mice with no immune system; triggering IHH expression within the body shouldn't trigger an immune response] induces ectopic cartilage and bone formation. Ectopic tissues derived largely if not exclusively from host cells. Treatment of clonal prechondrogenic RMD-1 and ATDC5 cells in culture with Ihh[The cell lines were already prochondrogenic] or recombinant amino half of Shh (recombinant N-terminal portion of Shh [rShh-N]) induced their differentiation into chondrocytes. Induction of RMD-1 cell differentiation by Ihh or rShh-N was synergistically enhanced by cotreatment with bone morphogenetic protein 2 (BMP-2) but was blocked by cotreatment with fibroblast growth factor 2 (FGF-2)[interesting considering in another study FGF-2 was used in the serum to make the cells prechondrogenic]. ."
In the study, The impaired bone anabolic effect of PTH in the absence of endogenous FGF2 is partially due to reduced ATF4 expression., FGF-2, which LSJL upregulated, increased ATF4 expression so perhaps in this study FGF-2 pushed ATF4 expression above equilibrium but in the other it didn't.
"FGF-4 blocks the induction of chondrogenesis by BMP-4"
Phenoytoin[prescription only] may be a way to increase Ihh levels via ATF4 as well:
Phenytoin stimulates chondrogenic differentiation in mouse clonal chondrogenic EC cells, ATDC5.
"We evaluated the effects of DPH(Phenytoin) on cartilage formation in a model system using ATDC5 cells, a clonal murine chondrogenic cell line. The ATDC5 cells demonstrated enhanced cartilage formation in cultures with DPH. During promoted chondrogenic differentiation, DPH increased the mRNA expression of TGF-beta1, BMP-4, Ihh, and Ptc, in a dose-dependent manner on Days 5 to 15[other epileptic drugs did not have this effect]."
DPH shares a common receptor with glucocorticoids. DPH may also interact with the thyroid receptor.
According to Inactivation of Patched1 in the mouse limb has novel inhibitory effects on the chondrogenic program., continuous activation of Ihh by inhibiting the hedgehog receptor Ptc-1 inhibited chondrogenesis so there is likely an equilibrium level of Ihh for chondrogenesis.
"a certain level of hedgehog signaling is required for optimal chondrogenesis, pathway activation can lead to inhibition of chondrogenesis in certain contexts."
In the study, The impaired bone anabolic effect of PTH in the absence of endogenous FGF2 is partially due to reduced ATF4 expression., FGF-2, which LSJL upregulated, increased ATF4 expression so perhaps in this study FGF-2 pushed ATF4 expression above equilibrium but in the other it didn't.
"FGF-4 blocks the induction of chondrogenesis by BMP-4"
Phenoytoin[prescription only] may be a way to increase Ihh levels via ATF4 as well:
Phenytoin stimulates chondrogenic differentiation in mouse clonal chondrogenic EC cells, ATDC5.
"We evaluated the effects of DPH(Phenytoin) on cartilage formation in a model system using ATDC5 cells, a clonal murine chondrogenic cell line. The ATDC5 cells demonstrated enhanced cartilage formation in cultures with DPH. During promoted chondrogenic differentiation, DPH increased the mRNA expression of TGF-beta1, BMP-4, Ihh, and Ptc, in a dose-dependent manner on Days 5 to 15[other epileptic drugs did not have this effect]."
DPH shares a common receptor with glucocorticoids. DPH may also interact with the thyroid receptor.
According to Inactivation of Patched1 in the mouse limb has novel inhibitory effects on the chondrogenic program., continuous activation of Ihh by inhibiting the hedgehog receptor Ptc-1 inhibited chondrogenesis so there is likely an equilibrium level of Ihh for chondrogenesis.
"a certain level of hedgehog signaling is required for optimal chondrogenesis, pathway activation can lead to inhibition of chondrogenesis in certain contexts."
According to Activating transcription factor 4., hypoxia also upregulates ATF4 so salubrinal may serve to get around a possible inability to generate hypoxia in adult bone.
Transcriptional regulation of bone formation.
"Major transcription factors including Sox9, Runx2, and Osterix, which are active in chondrocytes or osteoblasts or even in both cells, are indispensable for chondrocyte and/or osteoblast differentiation"
"Sox9-overexpressed mice in chondrocytes display chondrodysplasia by the inhibition of chondrocyte proliferation"<-too much Sox9 inhibits Cyclin D1 unless the Sox9 is methylated by CARM1.
The study notes that ATF4 is mainly expressed by osteoblasts.
"Major transcription factors including Sox9, Runx2, and Osterix, which are active in chondrocytes or osteoblasts or even in both cells, are indispensable for chondrocyte and/or osteoblast differentiation"
"Sox9-overexpressed mice in chondrocytes display chondrodysplasia by the inhibition of chondrocyte proliferation"<-too much Sox9 inhibits Cyclin D1 unless the Sox9 is methylated by CARM1.
The study notes that ATF4 is mainly expressed by osteoblasts.
"Investigations of the Atf4(-/-);Col2a1-Atf4 mouse model, in which Atf4 is selectively overexpressed in chondrocytes in an Atf4-null background, demonstrate that chondrocyte-derived Atf4 regulates osteogenesis during development and bone remodeling postnatally. Atf4 overexpression in chondrocytes of the Atf4(-/-);Col2a1-Atf4 double mutants corrects the reduction in stature and limb in Atf4(-/-) embryos and rectifies the decrease in Ihh expression, Hh signaling, proliferation and accelerated hypertrophy that characterize the Atf4(-/-) developing growth plate cartilages[So ATF4 only needs to be expressed in chondrocytes for bone to develop normally]. This genetic manipulation also restores the expression of osteoblastic marker genes, namely Ocn and bone sialoprotein, in Atf4(-/-) developing bones. In Atf4(-/-);Col2a1-Atf4 adult mice, all the defective bone parameters found in Atf4(-/-) mice, including bone volume, trabecular number and thickness, and bone formation rate, are rescued. In addition, the conditioned media of ex vivo cultures from wild-type or Atf4(-/-);Col2a1-Atf4, but not Atf4(-/-) cartilage, corrects the differentiation defects of Atf4(-/-) bone marrow stromal cells and Ihh-blocking antibody eliminates this effect."
If ATF4 based Ihh induction increased mesenchymal chondrogenesis then it wouldn't be likely that only stimulating ATF4 expression in chondrogenesis would rescue all symptoms of stature. Maybe, however that there's only a certain level of ATF4 required for Ihh to induce chondrogenesis and it didn't matter whether it was specifically in chondrocytes that the ATF4 was produced. Also, maybe Ihh can signal mesenchymal chondrogenesis even from chondrocytes as chondrocytic ATF4 can signal osteoblast differentiation too.
If ATF4 based Ihh induction increased mesenchymal chondrogenesis then it wouldn't be likely that only stimulating ATF4 expression in chondrogenesis would rescue all symptoms of stature. Maybe, however that there's only a certain level of ATF4 required for Ihh to induce chondrogenesis and it didn't matter whether it was specifically in chondrocytes that the ATF4 was produced. Also, maybe Ihh can signal mesenchymal chondrogenesis even from chondrocytes as chondrocytic ATF4 can signal osteoblast differentiation too.
According to Combinatorial control of ATF4-dependent gene transcription in osteoblasts., dimerization with the CEPB family occurs.
Positive regulation by GABAB receptor subunit-1 of chondrogenesis through acceleration of nuclear translocation of activating transcription factor-4.
"In cultured murine costal chondrocytes, mRNA was constitutively expressed for metabotropic GABA(B) receptor subunit-1 (GABA(B)R1), but not for GABA(B)R2. The predominant expression of GABA(B)R1 by prehypertrophic to hypertrophic chondrocytes in tibial sections of newborn mice. The GABA(B)R agonist baclofen failed to significantly affect chondrocytic differentiation while newborn mice knocked out of GABA(B)R1 (KO) showed a decreased body size[length of long bones was significantly shorter whereas short bones were about the same size] and delayed calcification in hyoid bone and forelimb and hindlimb digits. Delayed calcification was also seen in cultured metatarsals from KO mice with a marked reduction of Indian hedgehog (Ihh) expression. Introduction of GABA(B)R1 led to synergistic promotion of the transcriptional activity of activating transcription factor-4 (ATF4) essential for normal chondrogenesis, in addition to facilitating ATF4-dependent Ihh promoter activation. Although immunoreactive ATF4 was negligibly detected in the nucleus of chondrocytes from KO mice, ATF4 expression was again seen in the nucleus and cytoplasm after the retroviral introduction of GABA(B)R1 into cultured chondrocytes from KO mice. In nuclear extracts of KO chondrocytes, a marked decrease was seen in ATF4 DNA binding. GABA(B)R1 positively regulates chondrogenesis through a mechanism relevant to the acceleration of nuclear translocation of ATF4 for Ihh expression in chondrocytes."
So ATF4 may affect height through a GABABR1 and Ihh related mechanism.
"GABABR1 subunit [could] play a role in chondrogenesis through upregulation of Ihh expression"
The eIF2 kinase PERK and the integrated stress response facilitate activation of ATF6 during endoplasmic reticulum stress., states ATF4 is involved in ATF6 activation and that ATF6 deletion does not result in any changes in mouse phenotype.
"Vimentin{LSJL upregulates Vimentin}, a leucine zipper-containing intermediate filament protein, suppresses ATF4-dependent osteocalcin (Ocn){LSJL upregulates Ocn below 2-fold} transcription and osteoblast differentiation. TGFβ inhibits ATF4-dependent activation of Ocn by upregulation of vimentin expression. Osteoblasts lacking Atf4 (Atf4-/-) were less sensitive than wild-type (WT) cells to the inhibition by TGFβ on alkaline phosphatase activity, Ocn transcription and mineralization. The anabolic effect of a monoclonal antibody neutralizing active TGFβ ligands on WT mice was blunted in Atf4-/- mice. ATF4 is required for TGFβ-related suppression of Ocn transcription and osteoblast differentiation in vitro and in vivo. TGFβ did not directly regulate the expression of ATF4; instead, it enhanced the expression of vimentin, a negative regulator of ATF4, at the post-transcriptional level.
Knockdown of endogenous vimentin in 2T3 osteoblasts abolished the inhibition of Ocn transcription by TGFβ, confirming an indirect mechanism by which TGFβ acts through vimentin to suppress ATF4-dependent Ocn activation. Inhibition of PI3K/Akt/mTOR signaling, but not canonical Smad signaling, downstream of TGFβ, blocked TGFβ-induced synthesis of vimentin and inhibited ATF4-dependent Ocn transcription in osteoblasts. TGFβ stimulates vimentin production via PI3K-Akt-mTOR signaling, which leads to suppression of ATF4-dependent Ocn transcription and osteoblast differentiation."
Knockdown of endogenous vimentin in 2T3 osteoblasts abolished the inhibition of Ocn transcription by TGFβ, confirming an indirect mechanism by which TGFβ acts through vimentin to suppress ATF4-dependent Ocn activation. Inhibition of PI3K/Akt/mTOR signaling, but not canonical Smad signaling, downstream of TGFβ, blocked TGFβ-induced synthesis of vimentin and inhibited ATF4-dependent Ocn transcription in osteoblasts. TGFβ stimulates vimentin production via PI3K-Akt-mTOR signaling, which leads to suppression of ATF4-dependent Ocn transcription and osteoblast differentiation."
"TGFβ targets Runx2 via a canonical Smad signaling pathway to achieve its inhibition of both Runx2 and Ocn transcription, thereby suppressing osteoblast differentiation"
"Wortmannin, an inhibitor of Akt phosphorylation by PI3K; rapamycin (1 and 5 nM), an inhibitor of S6K phosphorylation by mTOR; and SB505124, an inhibitor of Smad2/3 phosphorylation downstream of the type I TGFβ receptor, were used to treat ROS17/2.8 cells."
"Both wortmannin and rapamycin (1 and 5 nM) drastically reduced vimentin protein expression induced by TGFβ, which correlated with a strong decrease in Akt and S6K phosphorylation." LSJL increases Akt phosphorylation.
"SB505124 (0.1 and 0.2 nM) effectively decreased TGFβ-induced Smad2 phosphorylation but not TGFβ-induced vimentin expression"
"silencing endogenous vimentin by siRNA-Vim attenuated the effects of TGFβ"
Positive regulation by γ-aminobutyric acid B receptor subunit-1 of chondrogenesis through acceleration of nuclear translocation of activating transcription factor-4.
y-aminobutyric acid is also known as GABA.
"In cultured murine costal chondrocytes, mRNA was constitutively expressed for metabotropic GABA(B) receptor subunit-1 (GABA(B)R1), but not for GABA(B)R2. Immunohistochemical analysis revealed the predominant expression of GABA(B)R1 by prehypertrophic to hypertrophic chondrocytes in tibial sections of newborn mice. The GABA(B)R agonist baclofen failed to significantly affect chondrocytic differentiation determined by Alcian blue staining and alkaline phosphatase activity in cultured chondrocytes, whereas newborn mice knocked out of GABA(B)R1 (KO) showed a decreased body size and delayed calcification in hyoid bone and forelimb and hindlimb digits. Delayed calcification was also seen in cultured metatarsals from KO mice with a marked reduction of Indian hedgehog gene (Ihh) expression. Introduction of GABA(B)R1 led to synergistic promotion of the transcriptional activity of activating transcription factor-4 (ATF4) essential for normal chondrogenesis, in addition to facilitating ATF4-dependent Ihh promoter activation. Although immunoreactive ATF4 was negligibly detected in the nucleus of chondrocytes from KO mice, ATF4 expression was again seen in the nucleus and cytoplasm after the retroviral introduction of GABA(B)R1 into cultured chondrocytes from KO mice. In nuclear extracts of KO chondrocytes, a marked decrease was seen in ATF4 DNA binding. GABA(B)R1 positively regulates chondrogenesis through a mechanism relevant to the acceleration of nuclear translocation of ATF4 for Ihh expression in chondrocytes."
"The introduction of ATF4 alone significantly increased the luciferase activity of a reporter vector containing six tandem copies of ATF4 binding site (6XOSE1) and Ihh promoter fragment"
"Abnormal endochondral bone formation and mineralization are shown in mice conditionally deleted of Ihh from Col2a1-expressing cells"
"GABABR1 subunit alone is constitutively expressed by chondrocytes to positively regulate chondrogenesis during skeletal development after acceleration of nuclear translocation of ATF4 toward up-regulation of Ihh expression in a manner irrelevant to the role as a dimeric partner for the canonical functional GABABR composed of both GABABR1 and GABABR2 subunits."
Positive regulation by γ-aminobutyric acid B receptor subunit-1 of chondrogenesis through acceleration of nuclear translocation of activating transcription factor-4.
y-aminobutyric acid is also known as GABA.
"In cultured murine costal chondrocytes, mRNA was constitutively expressed for metabotropic GABA(B) receptor subunit-1 (GABA(B)R1), but not for GABA(B)R2. Immunohistochemical analysis revealed the predominant expression of GABA(B)R1 by prehypertrophic to hypertrophic chondrocytes in tibial sections of newborn mice. The GABA(B)R agonist baclofen failed to significantly affect chondrocytic differentiation determined by Alcian blue staining and alkaline phosphatase activity in cultured chondrocytes, whereas newborn mice knocked out of GABA(B)R1 (KO) showed a decreased body size and delayed calcification in hyoid bone and forelimb and hindlimb digits. Delayed calcification was also seen in cultured metatarsals from KO mice with a marked reduction of Indian hedgehog gene (Ihh) expression. Introduction of GABA(B)R1 led to synergistic promotion of the transcriptional activity of activating transcription factor-4 (ATF4) essential for normal chondrogenesis, in addition to facilitating ATF4-dependent Ihh promoter activation. Although immunoreactive ATF4 was negligibly detected in the nucleus of chondrocytes from KO mice, ATF4 expression was again seen in the nucleus and cytoplasm after the retroviral introduction of GABA(B)R1 into cultured chondrocytes from KO mice. In nuclear extracts of KO chondrocytes, a marked decrease was seen in ATF4 DNA binding. GABA(B)R1 positively regulates chondrogenesis through a mechanism relevant to the acceleration of nuclear translocation of ATF4 for Ihh expression in chondrocytes."
"The introduction of ATF4 alone significantly increased the luciferase activity of a reporter vector containing six tandem copies of ATF4 binding site (6XOSE1) and Ihh promoter fragment"
"Abnormal endochondral bone formation and mineralization are shown in mice conditionally deleted of Ihh from Col2a1-expressing cells"
"GABABR1 subunit alone is constitutively expressed by chondrocytes to positively regulate chondrogenesis during skeletal development after acceleration of nuclear translocation of ATF4 toward up-regulation of Ihh expression in a manner irrelevant to the role as a dimeric partner for the canonical functional GABABR composed of both GABABR1 and GABABR2 subunits."
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