How proper sulfation can increase height

Previously, we've explored how genetic and protein changes can modify height like chromatin folding, DNA methylation, telomere length, and phosphorylation.  Sulfation is another way to modify genes and a potential way to modify genes involved in height growth.

We learned about Sulfation also in the articles about Lithium and FGF.  We learned that proteoglycan desulfation helped determine the extent of FGF signaling and the efficiency of chondrocyte autophagy.

Matrix disruptions, growth and degradation of cartilage with impaired sulfation.

"Diastrophic dysplasia (DTD) is an incurable recessive chondrodysplasia caused by mutations in SLC26A2 transporter responsible for sulfate uptake by chondrocytes[proper sulfate uptake is important for height growth]. The mutations cause undersulfation of glycosaminoglycans in cartilage. Studies of dtd mice with a knock-in Slc26a2 mutation showed an unusual progression of the disorder: net undersulfation is mild and normalizing with age, but the articular cartilage degrades with age and bones develop abnormally.We studied newborn dtd mice. Chondroitin sulfation across the proximal femur cartilage varied dramatically in dtd, but not in the wild type. Corresponding undersulfation of dtd was mild in most regions, but strong in narrow articular and growth plate regions crucial for bone development. This undersulfation correlated with chondroitin synthesis{sulfation increases chondroitin synthesis} rate measured via radioactive sulfate incorporation, explaining the sulfation normalization with age. Collagen orientation was reduced, and the reduction correlated with chondroitin undersulfation. Such disorientation involved the layer of collagen covering the articular surface and protecting cartilage from degradation. Malformation of this layer may contribute to the degradation progression with age and to collagen and proteoglycan depletion from the articular region, which we observed in mice already at birth."

"Sulfated molecular species like glycosaminoglycan (GAG) chains of proteoglycans, cholesterol, lipids, xenobiotics etc. provide numerous structural and signaling functions vital  for many tissues. In cartilage, sulfated GAGs provide tissue swelling and elasticity requiring sulfated GAGs in enormous quantities."<-so increasing the number of sulfated GAGs may increase tissue swelling and elasticity which may have an impact on height.

"The [mutation that causes chondrodysplasia] in the SLC26A2 sulfate/chloride antiporter (also called DTD sulfate transporter) [is] responsible for inorganic sulfate uptake by chondrocytes."

"In chondrocytes, SLC26A2 sulfate/chloride antiporter is the main source of inorganic sulfate. 

Most of this sulfate is spent to sulfate chondroitin which is then secreted to constitute ~90% of cartilage extracellular GAGs."<-So SLC26A2 sulfate/chloride antiporter is very important for height growth.  Anything that transports sulfate as well may also help increase height.

"Whether elevated chondroitin synthesis is also a driving force for chondrocyte enlargement  is unclear. Static hydration pressure of chondroitin sulfate proteoglycans (which swell matrix around chondrocytes) does not seem [to be the clear cause of chondrocyte enlargement]: Chondroitin sulfate density and, hence, hydration pressure at the columnar zone are similar to those at the enlargement and hypertrophic zones, but the columnar chondrocytes do not enlarge. However, we cannot completely exclude a second force due to dynamic hydration pressure from sulfated proteoglycans secreted and transiently concentrated near cells because our measurements cannot resolve pericellular matrix. If this force contributes, the nearly 2-fold reduction in chondroitin sulfation, believed to be the main cause of the pressure, is insufficient to appreciably reduce enlargement in the dtd growth plate region."

"The third hypothesized force driving cell enlargement is exerted from within chondrocytes by synthesizing intracellular components and uptaking water. This force does not seem to have substantial contribution from the elevated chondrotin sulfate synthesis because sulfation occurs in Golgi whose volume is small and does 

not augment. However, elevated synthesis of matrix proteins may contribute indirectly because it is accompanied by ~2.5-fold volumetric augmentation of the synthetic apparatus, endoplasmic reticulum, whose volume fraction per cell is substantial, ~15%"<-So it's unclear whether chondroitin synthesis increases chondrocyte enlargement which would be a potential factor for height increase.

LSJL may increase height in active growth plates by generating interstitial fluid flow which increases uptake of sulfate.  In the LSJL histology slides, we saw a greater presence of chondrocyte cells.  This greater number of cells could be caused by an increase in chondroitin sulfation due to IFF-driven sulfate transport.  The greater number and size of cells could be caused by a increase in matrix protecting chondrocytes from degradation and to sulfation increasing peak chondrocyte hypertrophy.

Now this still does not preclude LSJL being able to cause adult height growth as there have been numerous studies suggesting that hydrostatic pressure can induce chondrogenesis in MSCs.  However, it is possible that the height increase in rats was due to more sulfate transport rather than due to chondrogenic differentiation of MSCs.  What would have to be done is to perform LSJL Slc26A2 knockout mice versus wild type and compare load-driven bone lengthening between the two.  If Slc26A2 still increase height then you know that hydrostatic pressure driven chondrogenesis has an impact.

Expression patterns of sulfatase genes in the developing mouse embryo.

Sulfatase detected in cartilage regions:

Arsi(Cartilage)-up in LSJL

Arsj(cartilage)

SULF1(cartilage)-up in LSJL

SULF2(cartilage)

"Proliferating chondrocytes express Sulf2, whereas prehypertrophic chondrocytes express ArsI and hypertrophic chondrocytes express ArsI and Sulf1"

ARSB and GNS are also expressed in the bone.
Extracellular sulfatases support cartilage homeostasis by regulating BMP and FGF signaling pathways.

"Extracellular heparan endosulfatases Sulf-1 and Sulf-2 (Sulfs)are overexpressed in OA cartilage. Sulfs [regulate] bone morphogenetic protein (BMP)/Smad and fibroblast growth factor (FGF)/Erk signaling in articular cartilage. Spontaneous cartilage degeneration and surgically induced OA were significantly more severe in Sulf-1(-/-) and Sulf-2(-/-) mice compared with wild-type mice. MMP-13, ADAMTS-5, and the BMP antagonist noggin were elevated whereas col2a1 and aggrecan were reduced in cartilage and chondrocytes from Sulf(-/-) mice. Articular cartilage and cultured chondrocytes from Sulf(-/-) mice showed reduced Smad1 protein expression and Smad1/5 phosphorylation, whereas Erk1/2 phosphorylation was increased. In human chondrocytes, Sulfs siRNA reduced Smad phosphorylation but enhanced FGF-2-induced Erk1/2 signaling. Sulfs simultaneously enhance BMP but inhibit FGF signaling in chondrocytes and maintain cartilage homeostasis. "

"BMP-7 binding to the BMP receptor type IB leads to receptor dimerization so that the type I receptor phosphorylates Smad1/5/8. The BMP antagonist noggin is a secreted protein that interacts with heparan sulfate proteoglycans (HSPGs) at the cell membrane where it binds and prevents BMP-2, -4, -6, and -7 from activating their receptors"

"spontaneous cartilage degeneration [occurred] in Sulf−/− mice"

"BMP-7 stimulation increased col2a1, aggrecan, and noggin expression in Sulf−/− cells compared with the WT control, these levels were still lower than in WT chondrocytes stimulated with BMP-7"

"FGF-2 increased mmp-13 in both WT and Sulfs−/− cells, and especially Sulf-1−/− had the highest basal mmp-13. Thus, Sulf deficiency is associated with increased mmp-13 and noggin expression whereas col2a1 and aggrecan are reduced. Furthermore ADAMTS-5, a major aggrecan-degrading enzyme in cartilage, was overexpressed in Sulfs−/− compared with WT chondrocytes"

"Erk1/2 phosphorylation in Sulf−/− mice was markedly increased compared with that in WT"

"[There were] reduced basal levels of Smad1 protein expression and phosphorylation in Sulf-1−/− [mice when] compared with WT."

"Sulf-1 and Sulf-2 siRNA enhanced FGF-2-induced Erk1/2 phosphorylation by >100-fold compared with control siRNA"

The effect of desulfation of chondroitin sulfate on interactions with positively charged growth factors and upregulation of cartilaginous markers in encapsulated MSCs.

"Sulfated glycosaminoglycans (GAGs) are known to interact electrostatically with positively charged growth factors to modulate signaling.  Chondroitin sulfate (CS) was first desulfated to form chondroitin, and resulting crosslinked CS and chondroitin hydrogels were examined in vitro for release of positively charged model protein (histone) and for their effect on cartilaginous differentiation of encapsulated human mesenchymal stem cells (MSCs). Desulfation significantly increased the release of histone from chondroitin hydrogels (30.6 ± 2.3 μg released over 8 days, compared to natively sulfated CS with 20.2 ± 0.8 μg), suggesting that sulfation alone plays a significant role in modulating protein interactions with GAG hydrogels. MSCs in chondroitin hydrogels significantly upregulated gene expression of collagen II and aggrecan by day 21 in chondrogenic medium (115 ± 100 and 23.1 ± 7.9 fold upregulation of collagen II and aggrecan, respectively), compared to CS hydrogels and PEG-based swelling controls, indicating that desulfation may actually enhance the response of MSCs to soluble chondrogenic cues, such as TGF-β1."



Sulfated versus desulfated chondroitin.

"CS disaccharides were approximately 57.8% 6-sulfated and 26.8% 4-sulfated, as well as 1.3% nonsulfated"<-The desulfated version contains 98% desulfated version.

"100% CS-MA materials were found to exhibit greater retention of histone, while decreasing the CS content (with a reduction in sulfation and negative charge) resulted in significantly greater release"

So Sulfated Chondroitin retains more histone.

"hyaluronan, the unsulfated GAG has a similar chemical structure to desulfated chondroitin"